Regulators of biofilm formation and uses thereof

ABSTRACT

This invention relates to nucleic acid and amino acid sequences of genes regulating bacterial biofilm formation and to the use of these sequences as targets in the diagnosis, treatment, and prevention of bacterial infection and pathogenesis. In addition, the invention relates to screening methods for identifying modulators of bacterial biofilm formation and the development of antibacterial treatments.

This application claims benefit of U.S. provisional application60/303,286 and 60/373,233, filed Jul. 6, 2001 and Apr. 16, 2002,respectively. The disclosures of which are hereby incorporated byreference.

BACKGROUND OF THE INVENTION

This invention relates to nucleic acid and amino acid sequences of genesregulating bacterial biofilm formation and to the use of these sequencesas targets in the diagnosis, treatment, and prevention of bacterialinfection and pathogenesis. In addition, the invention relates toscreening methods for identifying modulators of bacterial biofilmformation and the development of antibacterial treatments.

Bacteria possess the ability to form aggregated, organized, colonialcommunities called biofilms. Distinct from their free-living planktoniccounterparts, bacterial cells that form biofilms secrete anexopolysacharide slime that surrounds and protects the bacterial colony.By adhering to each other and to surfaces or interfaces, thesematrix-enclosed bacterial populations can cause any number of problems.By attaching to a variety of surfaces such as contact lenses, waterpipes, hip replacements and food packaging, they can cause irritation,disease, immune rejection, and food poisoning.

In addition to attaching to abiotic surfaces, many biofilm-formingbacteria colonize living tissue where they cause serious infection. Forexample, Pseudomonas aeruginosa colonizes the lungs of cystic fibrosis(CF) patients as a biofilm. Chronic colonization of the airways by thisbacterial pathogen leads to debilitating exacerbation of pulmonaryinfection and constitutes the major cause of morbidity and mortality inCF populations. Colonization of the CF lung by P. aeruginosa generallypersists despite the use of long-term antibiotic therapy, sinceantibiotic treatment rarely results in complete eradication of theinfection.

As current antibiotic therapies offer limited effectiveness in treatingbiofilm infection, a need exists for developing therapeutic agents thatprevent biofilm formation. The discovery of polypeptides that regulatebiofilm formation and polynucleotides encoding such polypeptidesfulfills a need in the art by providing new compositions that are usefulin the diagnosis, treatment, and prevention of bacterial infection andpathogenesis, as well as biofilm formation in both industrial andmedical settings.

SUMMARY OF THE INVENTION

As is described in more detail below, we have discovered a regulatorysystem that modulates microbial phenotypic switching. In one aspect, theinvention features an isolated polypeptide that includes an amino acidsequence that is at least 50% (and preferably 60%, 65%, 70%, 75%, 80%,85%, 90%, or 95-99%) identical to the amino acid sequence of PvrR (SEQID NO:2), wherein expression of the polypeptide, in a microorganism,affects phenotype-mediated antibiotic-resistance in the microorganism.In preferred embodiments, the polypeptide includes the amino acidsequence of PvrR (SEQ ID NO:2) or consists essentially of the amino acidsequence of PvrR (SEQ ID NO:2) or a fragment thereof.

In a related aspect, the invention features an isolated polypeptidefragment of an isolated polypeptide that includes an amino acid sequencehaving at least 50% identity to the amino acid sequence of PvrR (SEQ IDNO:2). In preferred embodiments, such a polypeptide fragment includes atleast 300 contiguous amino acid residues of the amino acid sequence ofPvrR (SEQ ID NO:2). In other embodiments, the fragment is at least 250amino acid residues, 200 amino acid residues, or 100 amino acid residuesof the amino acid sequence of PvrR (SEQ ID NO:2).

In another aspect, the invention features an isolated polynucleotidehaving at least 50% identity to the nucleotide sequence of pvrR (SEQ IDNO:1), wherein expression of the polynucleotide, in a microorganism,affects phenotype-mediated antibiotic-resistance in the microorganism.In preferred embodiments, the isolated polynucleotide includes thenucleotide sequence of pvrR (SEQ ID NO:1) or a complement thereof. Inyet other preferred embodiments, the polynucleotide consists essentiallyof the nucleotide sequence of pvrR (SEQ ID NO:1) or a fragment thereof.

In still other related aspects, the invention features a vectorincluding any of the aforementioned isolated polynucleotides and a hostcell that includes the vector.

The invention further features a variety of screening assays foridentifying compounds that modulate phenotype-mediatedantibiotic-resistance, biofilm formation, or biofilm-mediated antibioticresistance. For example, the invention features a screening method thatis useful for identifying a compound that modulates the gene expressionof a regulator polynucleotide that affects phenotype-mediatedantibiotic-resistance in a microorganism. Such a method includes thesteps of: (a) providing a microbial cell (e.g., Pseudomonas, Vibrio,Salmonella, or Staphylococcus) that includes a polynucleotide having atleast 50% identity to the nucleotide sequence of pvrR (SEQ ID NO:1) (ora nucleotide sequence that is substantially identical to pvrR), whereinexpression of the polynucleotide, in the microbial cell, affectsphenotype-mediated antibiotic-resistance in the microbial cell; (b)contacting the microbial cell with a compound; and (c) comparing thelevel of gene expression of the polynucleotide in the presence of thecompound with the level of gene expression in the absence of thecompound; wherein a measurable difference in gene expression indicatesthat the compound modulates gene expression of a regulatorpolynucleotide that affects phenotype-mediated antibiotic-resistance ina microorganism.

In preferred embodiments, the screening method identifies a compoundthat increases or decreases transcription of the regulatorpolynucleotide. In other embodiments, the screening method identifies acompound that increases or decreases translation of an mRNA transcribedfrom the regulator polynucleotide.

In other preferred embodiments, the microbial cell is a phenotypicvariant (e.g., a small colony variant) having increased biofilmformation. Preferably, the small colony variant is a small colonyvariant of Pseudomonas, Vibrio, Salmonella, or Staphylococcus. In stillother embodiments, the small colony variant is a rough small colonyvariant, for example, a rough small colony variant of Pseudomonas,Vibrio, Salmonella, or Staphylococcus. In a preferred embodiment, therough small colony variant is Pseudomonas aeruginosa PA14 RSCV.

In other preferred embodiments, the activity of the compound used in thescreening assay is dependent upon the presence of the pvrR gene (SEQ IDNO:1) or a functional equivalent thereof. For example, the identifiedcompound targets and interacts with the pvrR gene (SEQ ID NO:1) or afunctional equivalent thereof. In still other preferred embodiments, theexpression of the regulator polynucleotide mediates phenotypic switchingof the microbial cell in the presence of a high concentration of anantibiotic. In other preferred embodiments of the screening method, thepolypeptide is expressed using an isolated polynucleotide that expressesa polypeptide having an amino acid sequence having at least 50% identityto the amino acid sequence of PvrR (SEQ ID NO:2) or a fragment thereof.

In another aspect, the invention features a screening method foridentifying a compound that modulates an activity of a polypeptide thataffects phenotype-mediated antibiotic-resistance in a microorganism. Themethod, in general, includes the steps of: (a) providing a microbialcell expressing a polypeptide having at least 50% identity to the aminoacid sequence of PvrR (SEQ ID NO:2) (or a polypeptide that issubstantially identical to PvrR), wherein expression of the polypeptide,in the microbial cell, affects phenotype-mediated antibiotic-resistancein the microbial cell; (b) contacting the microbial cell with acompound; and (c) comparing an activity of the polypeptide in thepresence of the compound with the activity in the absence of thecompound; wherein a measurable difference in the activity indicates thatthe compound modulates the activity of the polypeptide that affectsphenotype-mediated antibiotic-resistance in a microorganism. Inpreferred embodiments, the screening method identifies a compound thatincreases or decreases the activity of the polypeptide. Comparison ofthe activity of the polypeptide includes a variety of standardbiochemical analyses including immunological assays.

In preferred embodiments, the microbial cell utilized in the screeningassay is a phenotypic variant (e.g., Pseudomonas aeruginosa PA14 RSCV)having increased biofilm formation relative to wild-type.

In other preferred embodiments, the regulator polypeptide is an isolatedpolypeptide that includes an amino acid sequence having at least 50%identity to the amino acid sequence of PvrR (SEQ ID NO:2) (or apolypeptide that is substantially identical to PvrR). In particular,such a polypeptide has the ability to regulate phenotypic switching; toregulate biofilm-mediated antibiotic-resistance; to mediate phenotypicswitching of the microbial cell in the presence of a high concentrationof an antibiotic; or to affect susceptibility of the microbial cell toantibiotic treatment; or to regulate, or mediate, or affect, or anycombination of the aforementioned activities thereof. In other preferredembodiments, the regulator polypeptide is an element of a two-componentregulatory system. In yet other preferred embodiments, the polypeptideis expressed by an isolated polynucleotide having at least 50% identityto the nucleotide sequence of pvrR (SEQ ID NO:1) or a fragment thereof.

Typically, the activity of the compound identified in the screeningassay is dependent upon the presence of the PvrR polypeptide (SEQ IDNO:2) or a functional equivalent thereof. In particular aspects of thescreening assay, the compound targets the PvrR polypeptide (SEQ ID NO:2)or a functional equivalent thereof.

In another aspect, the invention features a screening method foridentifying a compound that modulates microbial biofilm formation. Thismethod, in general, includes the steps of: (a) culturing a microbialcell (e.g., Pseudomonas, Vibrio, Salmonella, or Staphylococcus) thatincludes a polypeptide having at least 50% identity to the amino acidsequence of PvrR (SEQ ID NO:2) (or a polypeptide that is substantiallyidentical to PvrR), wherein the microbial cell, upon culturing, forms abiofilm; (b) contacting the microbial cell with a compound; and (c)comparing microbial biofilm formation in the presence of the compoundwith microbial biofilm formation in the absence of the compound; whereina measurable difference in the microbial biofilm formation indicatesthat the compound modulates biofilm formation.

In preferred embodiments, the screening method identifies a compoundthat increases or decreases biofilm formation. Typically, such biofilmformation is measured by using any standard method, for example, byassaying microbial aggregation (e.g., by using a microscope); using asalt aggregation test; or by using an attachment assay.

In preferred embodiments, the microbial cell is a phenotypic varianthaving increased biofilm formation when compared to its wild-type suchas a small colony variant of Pseudomonas, Vibrio, Salmonella, orStaphylococcus. In other preferred embodiments, the small colony variantis a rough small colony variant of Pseudomonas, Vibrio, or Salmonella.In a preferred embodiment, the rough small colony variant is Pseudomonasaeruginosa PA14 RSCV.

In yet other preferred embodiments, the activity of the compoundutilized in the screening assay is dependent upon the presence of PvrRpolypeptide (SEQ ID NO: 2) or a functional equivalent thereof. Forexample, the identified compound targets and interacts with the PvrRpolypeptide (SEQ ID NO:2) or a functional equivalent thereof, resultingin increasing or decreasing its functional activity.

In still another embodiment, the expression of the polypeptide mediatesphenotypic switching of the microbial cell in the presence of a highconcentration of an antibiotic.

In another embodiment, the polypeptide is an isolated polypeptide thatincludes an amino acid sequence having at least 50% identity to theamino acid sequence of PvrR (SEQ ID NO:2), wherein expression of thepolypeptide, in a microorganism, affects phenotype-mediatedantibiotic-resistance in the microorganism.

In still another aspect, the invention features a method of treating amicrobial infection involving a microorganism that forms a biofilm in amammal. The method, in general, includes administering to the mammal atherapeutically-effective amount of a compound that induces or repressesexpression or activity of a polypeptide that includes an amino acidsequence having at least 50% identity to the amino acid sequence of PvrR(SEQ ID NO:2) (or a polypeptide that is substantially identical to PvrR)or a fragment thereof, wherein expression of the polypeptide or thefragment thereof, in a microorganism, affects phenotype-mediatedantibiotic-resistance in the microorganism.

In preferred embodiments, the method further includes administering tothe mammal a therapeutically-effective amount of an antibiotic. Thetreatment is particularly useful for treating patients having cysticfibrosis or a chronic microbial infection or both. In other preferredembodiments, the microorganism treated using the method belongs to thegenus Pseudomonas, Vibrio, Salmonella, or Staphylococcus.

In yet another aspect, the invention features a method of cleaning,disinfecting, or decontaminating a surface at least partially covered bya microorganism that forms a biofilm, the method involving contactingthe microorganism with a cleaning composition including a compound thatinduces or represses expression or activity of a polypeptide thatincludes an amino acid sequence having at least 50% identity to theamino acid sequence of PvrR (SEQ ID NO:2) (or a polypeptide that issubstantially identical to PvrR) or fragment thereof, wherein expressionof the polypeptide or the fragment thereof, in a microorganism, affectsphenotype-mediated antibiotic-resistance in the microorganism.

In yet another aspect, the invention features a screening method foridentifying a compound that decreases pathogenicity of anantibiotic-resistant phenotypic variant. The method, in general,includes the steps of: (a) contacting an antibiotic-resistant phenotypicvariant with a candidate compound; and (b) measuring reversion of theantibiotic-resistant phenotypic variant to a wild-type phenotype, anincrease in reversion indicating that the compound decreasespathogenicity of the antibiotic-resistant phenotypic variant. Inpreferred embodiments, the antibiotic-resistant phenotypic variant iscultured in the absence of an antibiotic, has increased biofilmformation; is a rough small colony variant; is a hyperpiliated variant;has increased hydrophobicity; has an alteration in a surface component;or is a pathogen such as a Gram positive bacterium (e.g.,Staphylococcus) or a Gram negative bacterium (e.g., Vibrio, Pseudomonas,or Salmonella).

In another aspect, the invention features a screening method foridentifying a compound that decreases pathogenicity of anantibiotic-resistant phenotypic variant. The method, in general,includes the steps of: (a) culturing an antibiotic-resistant phenotypicvariant with a candidate compound in the presence of an antibiotic; and(b) comparing the number of antibiotic-resistant phenotypic variants inthe presence of the compound to the number of antibiotic-resistantphenotypic variants in the absence of the compound, a decrease in thenumber of the antibiotic-resistant phenotypic variants in the presenceof the compound indicating that the compound decreases pathogenicity ofthe antibiotic-resistant phenotypic variant.

In yet another aspect, the invention features a screening method foridentifying a polynucleotide encoding a regulator polypeptide, themethod including the steps of: (a) providing a mutagenized microbe; (b)culturing the mutagenized microbe in the presence of an antibiotic; and(c) comparing the mutagenized microbe with a control wild-type microbe,wherein a change in the number of phenotypic variants identifies themutagenized microbe as having a mutation in a polynucleotide encoding aregulator polypeptide. In preferred embodiments, the phenotypic variantis a small colony variant.

In another aspect, the invention features a screening method foridentifying a polynucleotide encoding a regulator polypeptide thatmodulates an antibiotic-resistant phenotype of a microorganism. Themethod, in general, includes the steps of: (a) identifying anantibiotic-resistant phenotypic variant of a microorganism including afirst phenotype; (b) mutagenizing the antibiotic-resistant phenotypicvariant of the microorganism, thereby generating a mutated phenotypicvariant of the microorganism; and (c) selecting the mutated phenotypicvariant of step (b) having a second phenotype, other than the firstphenotype of the antibiotic-resistant phenotypic variant, wherein thesecond phenotype identifies a mutation in the mutated phenotypic variantof step (b); and (d) using the mutation for identifying a polynucleotideencoding a regulator polypeptide that modulates an antibiotic-resistantphenotype of a microorganism. In preferred embodiments, the secondphenotype includes a wild-type phenotype.

In yet another aspect, the invention features a screening method foridentifying a polynucleotide encoding a regulator polypeptide thatmodulates phenotype-mediated antibiotic-resistance of a microorganism.The method, in general, includes the steps of: (a) transforming anantibiotic-resistant phenotypic variant of a microorganism with acandidate polynucleotide encoding a regulator polypeptide; and (b)culturing the transformed antibiotic-resistant phenotypic variant of amicroorganism under conditions suitable for expression of the regulatorpolypeptide; and (c) measuring reversion of the transformedantibiotic-resistant phenotypic variant of the microorganism to awild-type phenotype, an increase in reversion identifies thepolynucleotide as encoding a regulator polypeptide that modulatesphenotype-mediated antibiotic-resistance.

In preferred embodiments, the polynucleotide encodes a regulatorpolypeptide that modulates a phenotypic switch from anantibiotic-resistant phenotype to an antibiotic-susceptible phenotype.In other preferred embodiments, the candidate polynucleotide has atleast 50% identity to the nucleotide sequence of pvrR (SEQ ID NO:1) (ora polynucleotide sequence that is substantially identical to pvrR). Inother embodiments, the candidate polynucleotide sequence issubstantially identical to any one of the polynucleotides shown in FIGS.5B, 5C, 6A-6K, and 7A-7E. In other preferred embodiments, the candidatepolynucleotide encodes a polypeptide that is an element of atwo-component regulatory system.

In another aspect, the invention features an isolated polypeptideincluding an amino acid sequence that is substantially identical to theamino acid sequence of any one the polypeptides shown in FIGS. 5E (SEQID NO: 4) and 6L-6V (SEQ ID NOS: 19-29), each of which are encoded by apolynucleotide of the ORF1 region.

For example, with respect to the ORF1 region, the invention features anisolated polypeptide that includes an amino acid sequence that is atleast 50% (and preferably 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95-99%)identical to the amino acid sequence of the polypeptide shown in FIG. 5E(SEQ ID NO: 4) or to a polypeptide shown in FIGS. 6L-6V (SEQ ID NOS:19-29), wherein expression of the polypeptide, in a microorganism,affects phenotype-mediated antibiotic-resistance in the microorganism.Preferably, the polypeptide includes the amino acid sequence shown inFIG. 5E or consists essentially of the amino acid sequence shown in FIG.5E or a fragment thereof.

In a related aspect, the invention features an isolated polypeptidefragment of an isolated polypeptide that includes an amino acid sequencehaving at least 50% identity to the amino acid sequence the polypeptideshown in FIG. 5E or to a polypeptide shown in any one of FIGS. 6L-6V. Inpreferred embodiments, such a polypeptide fragment includes at least 400contiguous amino acid residues of the amino acid sequence shown in anyone of FIGS. 5E and 6L-6V. In other embodiments, the fragment is atleast 300 amino acid residues, 200 amino acid residues, or 100 aminoacid residues of the polypeptides shown in FIGS. 5E and 6L-6V.

In another aspect, the invention features an isolated polynucleotidemolecule including a sequence substantially identical to any one of thepolynucleotides shown in FIGS. 5B (SEQ ID NO:3) and 6A-6K (SEQ ID NOS:8-18), which are found in the ORF1 region. In preferred embodiments, theisolated polynucleotide molecule has at least 45%, 50%, 60%, 70%, 80%,90%, or even 95-99% identity to any one of these isolated molecules.

For example, with respect to the ORF1 region, the invention features anisolated polynucleotide having at least 50% identity to the nucleotidesequence shown in FIG. 5B or to any one of the nucleotide sequencesshown in FIGS. 6A-6K, wherein expression of the polynucleotide, in amicroorganism, affects phenotype-mediated antibiotic-resistance in themicroorganism. In preferred embodiments, the isolated polynucleotideincludes the nucleotide sequence shown in FIG. 5B or a complementthereof. In yet other preferred embodiments, the polynucleotide consistsessentially of the nucleotide sequence shown in FIG. 5B or a fragmentthereof.

In still other related aspects, the invention features a vectorincluding any of the aforementioned isolated polynucleotides and a hostcell that includes the vector.

The invention further features a variety of screening assays foridentifying compounds that modulate phenotype-mediatedantibiotic-resistance, biofilm formation, or biofilm-mediated antibioticresistance. For example, the invention features a screening method thatis useful for identifying a compound that modulates the gene expressionof a regulator polynucleotide that affects phenotype-mediatedantibiotic-resistance in a microorganism. Such a method includes thesteps of: (a) providing a microbial cell (e.g., Pseudomonas, Vibrio,Salmonella, or Staphylococcus) that includes a polynucleotide that issubstantially identical to any one of the nucleotide sequences shown inFIG. 5B or 6A-6K (or a polynucleotide having at least 40% identity toany one of these sequences), wherein expression of the polynucleotide,in the microbial cell, affects phenotype-mediated antibiotic-resistancein the microbial cell; (b) contacting the microbial cell with acompound; and (c) comparing the level of gene expression of thepolynucleotide in the presence of the compound with the level of geneexpression in the absence of the compound; wherein a measurabledifference in gene expression indicates that the compound modulates geneexpression of a regulator polynucleotide that affects phenotype-mediatedantibiotic-resistance in a microorganism.

In preferred embodiments, the screening method identifies a compoundthat increases or decreases transcription of the regulatorpolynucleotide. In other embodiments, the screening method identifies acompound that increases or decreases translation of an mRNA transcribedfrom the regulator polynucleotide.

In other preferred embodiments, the microbial cell is a phenotypicvariant (e.g., a small colony variant) having increased biofilmformation. Preferably, the small colony variant is a small colonyvariant of Pseudomonas, Vibrio, Salmonella, or Staphylococcus. In stillother embodiments, the small colony variant is a rough small colonyvariant, for example, a rough small colony variant of Pseudomonas,Vibrio, Salmonella, or Staphylococcus. In a preferred embodiment, therough small colony variant is Pseudomonas aeruginosa PA14 RSCV.

In other preferred embodiments, the activity of the compound used in thescreening assay is dependent upon the presence of any one of thepolynucleotides shown in FIG. 5B or 6A-6K, or a functional equivalentthereof. For example, the identified compound targets any one of thepolynucleotides shown in FIG. 5B or 6A-6K or a functional equivalentthereof. In still other preferred embodiments, the expression of theregulator polynucleotide mediates phenotypic switching of the microbialcell in the presence of a high concentration of an antibiotic. In otherpreferred embodiments of the screening method, the polypeptide isexpressed using an isolated polynucleotide that encodes a polypeptidethat is substantially identical to any one of the polynucleotides shownFIGS. 5B and 6A-6K or a fragment thereof.

In another aspect, the invention features a screening method foridentifying a compound that modulates an activity of a polypeptide thataffects phenotype-mediated antibiotic-resistance in a microorganism. Themethod, in general, includes the steps of: (a) providing a microbialcell expressing a polypeptide that is substantially identical to any oneof the polypeptides shown in FIGS. 5E and 6L-6V (or a polypeptide havingat least 40% identity to any one of these sequences), wherein expressionof the polypeptide, in the microbial cell, affects phenotype-mediatedantibiotic-resistance in the microbial cell; (b) contacting themicrobial cell with a compound; and (c) comparing an activity of thepolypeptide in the presence of the compound with the activity in theabsence of the compound; wherein a measurable difference in the activityindicates that the compound modulates the activity of the polypeptidethat affects phenotype-mediated antibiotic-resistance in amicroorganism. In preferred embodiments, the screening method identifiesa compound that increases or decreases the activity of the polypeptide.Comparison of the activity of the polypeptide includes a variety ofstandard biochemical analyses including immunological assays.

In preferred embodiments, the microbial cell utilized in the screeningassay is a phenotypic variant (e.g., Pseudomonas aeruginosa PA14 RSCV)having increased biofilm formation.

In other preferred embodiments, the regulator polypeptide is an isolatedpolypeptide that includes an amino acid sequence that is substantiallyidentical to any one of the polypeptides shown in FIGS. 5E and 6L-6V (ora polypeptide having at least 40% identity to any one of thesesequences). In particular, such a polypeptide has the ability toregulate phenotypic switching; to regulate biofilm-mediatedantibiotic-resistance; to mediate phenotypic switching of the microbialcell in the presence of a high concentration of an antibiotic; or toaffect susceptibility of the microbial cell to antibiotic treatment; orany combination thereof. In other preferred embodiments, the regulatorpolypeptide is an element of a two-component regulatory system. In yetother preferred embodiments, the polypeptide is expressed by an isolatedpolynucleotide that is substantially identical to any one of thenucleotide sequences shown in FIGS. 5B and 6A-6K (or a polynucleotidehaving at least 40% identity to any one of these sequences) or afragment thereof, upon which the activity of the regulator polypeptideis increased or decreased.

Typically, the activity of the compound identified in the screeningassay is dependent upon the presence of any one of the polypeptidesshown in FIGS. 5E and 6L-6V or a functional equivalent thereof. Inparticular aspects of the screening assay, the compound targets orinteracts with any one of the polypeptides shown in FIGS. 5E and 6L-6Vor a functional equivalent thereof.

In another aspect, the invention features a screening method foridentifying a compound that modulates microbial biofilm formation. Thismethod, in general, includes the steps of: (a) culturing a microbialcell (e.g., Pseudomonas, Vibrio, Salmonella, or Staphylococcus) thatincludes a polypeptide that is substantially identical to any one of thepolypeptides shown in FIGS. 5E and 6L-6V (or a polypeptide having atleast 40% identity to any one of these sequences), wherein the microbialcell, upon culturing, forms a biofilm; (b) contacting the microbial cellwith a compound; and (c) comparing microbial biofilm formation in thepresence of the compound with microbial biofilm formation in the absenceof the compound; wherein a measurable difference in the microbialbiofilm formation indicates that the compound modulates biofilmformation.

In preferred embodiments, the screening method identifies a compoundthat increases or decreases biofilm formation. Typically, such biofilmformation is measured by using any standard method, for example, byassaying microbial aggregation (e.g., by using a microscope); using asalt aggregation test; or by using an attachment assay.

In preferred embodiments, the microbial cell is a phenotypic varianthaving increased biofilm formation when compared to its wild-type suchas a small colony variant of Pseudomonas, Vibrio, Salmonella, orStaphylococcus. In other preferred embodiments, the small colony variantis a rough small colony variant of Pseudomonas, Vibrio, or Salmonella.

In yet other preferred embodiments, the activity of the compoundutilized in the screening assay is dependent upon the presence of thepolypeptide or a functional equivalent thereof. For example, theidentified compound targets or interacts with the polypeptide or afunctional equivalent thereof, resulting in increasing or decreasing itsfunctional activity.

In still another embodiment, the expression of the polypeptide mediatesphenotypic switching of the microbial cell in the presence of a highconcentration of an antibiotic.

In another embodiment, the polypeptide is an isolated polypeptide thatincludes an amino acid sequence that is substantially identical to anyone of the polypeptides shown in FIGS. 5E and 6L-6V (or a polypeptidehaving at least 40% identity to any one of these sequences), whereinexpression of the polypeptide, in a microorganism, affectsphenotype-mediated antibiotic-resistance in the microorganism.

In still another aspect, the invention features a method of treating amicrobial infection involving a microorganism that forms a biofilm in amammal. The method, in general, includes administering to the mammal atherapeutically-effective amount of a compound that induces or repressesexpression or activity of a polypeptide that includes a polypeptide thatis substantially identical to any one of the polypeptides shown in FIGS.5E and 6L-6V or a fragment thereof (or a polypeptide having at least 40%identity to any one of these sequences), wherein expression of thepolypeptide or the fragment thereof, in a microorganism, affectsphenotype-mediated antibiotic-resistance in the microorganism.

In another aspect, the invention features an isolated polypeptideincluding an amino acid sequence that is substantially identical to theamino acid sequence of any one of the polypeptides shown in FIG. 5F andFIGS. 7F-7J, each of which are encoded by a polynucleotide of the ORF3region.

For example, with respect to the ORF3 region, the invention features anisolated polypeptide that includes an amino acid sequence that is atleast 50% (and preferably 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95-99%)identical to the amino acid sequence of any one of the polypeptidesshown in FIGS. 5F (SEQ ID NO:6) and 7F-7J (SEQ ID NOS:35-39), whereinexpression of the polypeptide, in a microorganism, affectsphenotype-mediated antibiotic-resistance in the microorganism.Preferably, the polypeptide includes the amino acid sequence shown inFIG. 7J (SEQ ID NO:39) or consists essentially of the amino acidsequence shown in FIGS. 5F (SEQ ID NO:6) and 7F-7I (SEQ ID NOS:35-38) ora fragment thereof.

In a related aspect, the invention features an isolated polypeptidefragment of an isolated polypeptide that includes an amino acid sequencehaving at least 50% identity to the amino acid sequence of thepolypeptides shown in FIGS. 5F and 7F-7J. In preferred embodiments, sucha polypeptide fragment includes at least 300 contiguous amino acidresidues of the amino acid sequence shown in any one of FIGS. 5F and7F-7J. In other embodiments, the fragment is at least 200 amino acidresidues, or 100 amino acid residues of the polypeptides shown in FIGS.5F and 7F-7J.

In another aspect the invention features an isolated polynucleotidemolecule including a sequence substantially identical to any one of thepolynucleotides shown in FIGS. 5C (SEQ ID NO:5) and 7A-7E (SEQ IDNOS:30-34). In preferred embodiments, the isolated polynucleotidemolecule has at least 45%, 50%, 60%, 70%, 80%, 90%, or even 95% identityto any one of these molecules.

For example with respect to the ORF3 region, the invention features anisolated polynucleotide having at least 50% identity to any one of thenucleotide sequences shown in FIGS. 5C and 7A-7E, wherein expression ofthe polynucleotide, in a microorganism, affects phenotype-mediatedantibiotic-resistance in the microorganism. In preferred embodiments,the isolated polynucleotide includes the nucleotide sequence shown inFIG. 5C or a complement thereof. In yet other preferred embodiments, thepolynucleotide consists essentially of the nucleotide sequence shown inFIG. 5C or a fragment thereof.

In still other related aspects, the invention features a vectorincluding any of the aforementioned isolated polynucleotides and a hostcell that includes the vector.

The invention further features a variety of screening assays foridentifying compounds that modulate phenotype-mediatedantibiotic-resistance, biofilm formation, or biofilm-mediated antibioticresistance. For example, the invention features a screening method thatis useful for identifying a compound that modulates the gene expressionof a regulator polynucleotide that affects phenotype-mediatedantibiotic-resistance in a microorganism. Such a method includes thesteps of: (a) providing a microbial cell (e.g., Pseudomonas, Vibrio,Salmonella, or Staphylococcus) that includes a polynucleotidesubstantially identical to the nucleotide sequences shown in FIGS. 5Cand 7A-7E (or a polynucleotide having at least 45% identity to any oneof these sequences), wherein expression of the polynucleotide, in themicrobial cell, affects phenotype-mediated antibiotic-resistance in themicrobial cell; (b) contacting the microbial cell with a compound; and(c) comparing the level of gene expression of the polynucleotide in thepresence of the compound with the level of gene expression in theabsence of the compound; wherein a measurable difference in geneexpression indicates that the compound modulates gene expression of aregulator polynucleotide that affects phenotype-mediatedantibiotic-resistance in a microorganism.

In preferred embodiments, the screening method identifies a compoundthat increases or decreases transcription of the regulatorpolynucleotide. In other embodiments, the screening method identifies acompound that increases or decreases translation of an mRNA transcribedfrom the regulator polynucleotide.

In other preferred embodiments, the microbial cell is a phenotypicvariant (e.g., a small colony variant) having increased biofilmformation. Preferably, the small colony variant is a small colonyvariant of Pseudomonas, Vibrio, Salmonella, or Staphylococcus. In stillother embodiments, the small colony variant is a rough small colonyvariant, for example, a rough small colony variant of Pseudomonas,Vibrio, Salmonella, or Staphylococcus. In a preferred embodiment, therough small colony variant is Pseudomonas aeruginosa PA14 RSCV.

In other preferred embodiments, the activity of the compound used in thescreening assay is dependent upon the presence of any one of thepolynucleotides shown in FIGS. 5C and 7A-7E or a functional equivalentthereof. For example, the identified compound targets or interacts withany one of the polynucleotides shown in FIGS. 5C and 7A-7E or afunctional equivalent thereof. In still other preferred embodiments, theexpression of the regulator polynucleotide mediates phenotypic switchingof the microbial cell in the presence of a high concentration of anantibiotic. In other preferred embodiments of the screening method, thepolypeptide is expressed from an isolated polynucleotide that expressesa polypeptide that includes an amino acid sequence having at least 50%identity to any one of the amino acid sequences shown in FIGS. 5F and7F-7J or a fragment thereof.

In another aspect, the invention features a screening method foridentifying a compound that modulates an activity of a polypeptide thataffects phenotype-mediated antibiotic-resistance in a microorganism. Themethod, in general, includes the steps of: (a) providing a microbialcell expressing a polypeptide that is substantially identical to any oneof the polypeptides shown in FIGS. 5F and 7F-7J (or a polypeptide havingat least 45% identity to any one of these sequences), wherein expressionof the polypeptide, in the microbial cell, affects phenotype-mediatedantibiotic-resistance in the microbial cell; (b) contacting themicrobial cell with a compound; and (c) comparing an activity of thepolypeptide in the presence of the compound with the activity in theabsence of the compound; wherein a measurable difference in the activityindicates that the compound modulates the activity of the polypeptidethat affects phenotype-mediated antibiotic-resistance in amicroorganism. In preferred embodiments, the screening method identifiesa compound that increases or decreases the activity of the polypeptide.Comparison of the activity of the polypeptide includes a variety ofstandard biochemical analyses including immunological assays.

In preferred embodiments, the microbial cell utilized in the screeningassay is a phenotypic variant (e.g., Pseudomonas aeruginosa PA14 RSCV)having increased biofilm formation.

In other preferred embodiments, the regulator polypeptide is an isolatedpolypeptide that includes an amino acid sequence that is substantiallyidentical to any one of the polypeptides shown in FIGS. 5F and 7F-7J (ora polypeptide having at least 45% identity to any one of thesesequences). In particular, such a polypeptide has the ability toregulate phenotypic switching; to regulate biofilm-mediatedantibiotic-resistance; to mediate phenotypic switching of the microbialcell in the presence of a high concentration of an antibiotic; or toaffect susceptibility of the microbial cell to antibiotic treatment; orany combination thereof. In other preferred embodiments, the regulatorpolypeptide is an element of a two-component regulatory system. In yetother preferred embodiments, the polypeptide is expressed by an isolatedpolynucleotide substantially identical to any one of the nucleotidesequences shown in FIGS. 5C and 7A-7E (or by a polynucleotide having atleast 45% identity to any one of these sequences) or a fragment thereof,upon which the activity of the regulator polypeptide is increased ordecreased.

Typically, the activity of the compound identified in the screeningassay is dependent upon the presence of any one of the polypeptidesshown in FIGS. 5F and 7F-7J or a functional equivalent thereof. Inparticular aspects of the screening assay, the compound targets andinteracts with the polypeptide of FIGS. 5F and 7F-7J or a functionalequivalent thereof.

In another aspect, the invention features a screening method foridentifying a compound that modulates microbial biofilm formation. Thismethod, in general, includes the steps of: (a) culturing a microbialcell (e.g., Pseudomonas, Vibrio, Salmonella, or Staphylococcus) thatincludes a polypeptide substantially identical to any one of the aminoacid sequences shown in FIGS. 5F and 7F-7J (or a polypeptide having atleast 45% identity to any one of these sequences), wherein the microbialcell, upon culturing, forms a biofilm; (b) contacting the microbial cellwith a compound; and (c) comparing microbial biofilm formation in thepresence of the compound with microbial biofilm formation in the absenceof the compound; wherein a measurable difference in the microbialbiofilm formation indicates that the compound modulates biofilmformation.

In preferred embodiments, the screening method identifies a compoundthat increases or decreases biofilm formation. Typically, such biofilmformation is measured by using any standard method, for example, byassaying microbial aggregation (e.g., by using a microscope); using asalt aggregation test; or by using an attachment assay.

In preferred embodiments, the microbial cell is a phenotypic varianthaving increased biofilm formation when compared to its wild-type suchas a small colony variant of Pseudomonas, Vibrio, Salmonella, orStaphylococcus. In other preferred embodiments, the small colony variantis a rough small colony variant of Pseudomonas, Vibrio, or Salmonella.

In yet other preferred embodiments, the activity of the compoundutilized in the screening assay is dependent upon the presence of thepolypeptide or a functional equivalent thereof. For example, theidentified compound targets and interacts with the polypeptide or afunctional equivalent thereof, resulting in increasing or decreasing itsfunctional activity.

In still another embodiment, the expression of the polypeptide mediatesphenotypic switching of the microbial cell in the presence of a highconcentration of an antibiotic.

In another embodiment, the polypeptide is an isolated polypeptide thatincludes an amino acid sequence that is substantially identical to anyone of the amino acid sequences shown in FIGS. 5F and 7F-7J (or apolypeptide having at least 45% identity to any one of these sequences),wherein expression of the polypeptide, in a microorganism, affectsphenotype-mediated antibiotic-resistance in the microorganism.

In still another aspect, the invention features a method of treating amicrobial infection involving a microorganism that forms a biofilm in amammal. The method, in general, includes administering to the mammal atherapeutically-effective amount of a compound that induces or repressesexpression or activity of a polypeptide that includes an amino acidsequence that is substantially identical to any one of the amino acidsequences shown in FIGS. 5F and 7F-7J or a fragment thereof (or apolypeptide having at least 45% identity to any one of these sequences),wherein expression of the polypeptide or the fragment thereof, in amicroorganism, affects phenotype-mediated antibiotic-resistance in themicroorganism.

In preferred embodiments, the method further includes administering tothe mammal a therapeutically-effective amount of an antibiotic. Thetreatment is particularly useful for treating patients having cysticfibrosis or a chronic infection or both. In other preferred embodiments,the microorganism treated using the method belongs to the genusPseudomonas, Vibrio, Salmonella, or Staphylococcus.

In yet another aspect, the invention features a method of cleaning,disinfecting, or decontaminating a surface at least partially covered bya microorganism that forms a biofilm, the method involving contactingthe microorganism with a cleaning composition including a compound thatinduces or represses expression or activity of a polypeptide thatincludes an amino acid sequence having at least 50% identity to theamino acid sequence of FIGS. 5E, 5F, 6L-6V, and 7F-7J or fragmentthereof (or a polypeptide that is substantially identical to any one ofthese polypeptides), wherein expression of the polypeptide or thefragment thereof, in a microorganism, affects phenotype-mediatedantibiotic-resistance in the microorganism.

The invention also features methods for identifying compounds useful fortreating a patient having a biofilm infection. The method includes thesteps of contacting a biofilm in vitro with (i) an antibiotic and (ii) acandidate compound (e.g., a compound that modulates the expression, atthe transcriptional, post-transcriptional, translational, orpost-translational levels, of a polynucleotide having at least 50%identity to any of the polynucleotides described herein (or that issubstantially identical to a polynucleotide described herein), anddetermining whether the biofilm grows more slowly than (a) biofilm cellscontacted with an antibiotic but not contacted with the test compound,and (b) biofilm cells contacted with the candidate compound but not withthe antibiotic. In another embodiment, the biofilm is contacted with twoor more different antibiotics. Exemplary antibiotics useful in themethod include amikacin, aminoglicosides (e.g., tobramycin), aztreonam,carbenicillin, cephalosporines (e.g., ceftazidime or cefipime),chloramphenicol, gentamicin, levofloxacin, meropenem, piperacillin,tazobactam, tetracycline, and quinolones (e.g., ciprofloxacin). Acandidate compound that reduces biofilm formation in the presence of anantibiotic (or combination of different antibiotics), but does notdecrease biofilm formation in the absence of the antibiotic (orcombination of different antibiotics), is a compound that is useful incombination therapy for treating a patient having a biofilm infection.

The invention further features a method for treating a patient having abiofilm infection, by administering to the patient an antibiofilmcombination therapy that includes a compound identified as modulatingexpression, at the transcriptional, post-transcriptional, translational,or post-translational levels, of a polynucleotide having at least 50%identity to any of the polynucleotides described herein (or that issubstantially identical to a polynucleotide described herein) and one ormore antibiotics, including, but not limited to, amikacin,aminoglicosides (e.g., tobramycin), aztreonam, carbenicillin,cephalosporines (e.g., ceftazidime or cefipime), chloramphenicol,gentamicin, levofloxacin, meropenem, piperacillin, tazobactam,tetracycline, and quinolones (e.g., ciprofloxacin), simultaneously orwithin a period of time (e.g., 14 to 21 days) sufficient to inhibit thegrowth of the biofilm.

Preferably, the compound and antibiotic are administered within fifteendays of each other, more preferably within five or ten days of eachother, and most preferably within twenty-four hours of each other oreven simultaneously. Exemplary biofilms treated according to any of themethods described herein are those formed by bacteria, including but notlimited to, Pseudomonas, Staphylococcus, Salmonella, Vibrio,Haemophilus, Mycobacterium, Helicobacter, Burkholderia, or Streptococci.

In a related aspect, the invention also features a method for treating apatient having a biofilm such as one formed from Pseudomonas (e.g.,Pseudomonas aeruginosa). In this method, a patient is administered (a) afirst compound (e.g., a compound that modulates the expression, at thetranscriptional, post-transcriptional, translational, orpost-translational; of a polynucleotide having at least 50% identity toa polynucleotide described herein (or that is substantially identical toa polynucleotide described herein)), and (b) one or more antibiotics(such as amikacin, aminoglicosides (e.g., tobramycin), aztreonam,carbenicillin, cephalosporines (e.g., ceftazidime or cefipime),chloramphenicol, gentamicin, levofloxacin, meropenem, piperacillin,tazobactam, tetracycline, and quinolones (e.g., ciprofloxacin). Ifdesired, the therapy includes administration of two antibioticsaccording to standard methods known in the art. Such dual antibioticcombinations most preferably include high-dose tobramycin plusmeropenem, meropenem plus ciprofloxacin, or tobramycin (4 μg/ml), orcefipime. Other preferred combinations include piperacillin plustazobactam, or piperacillin plus ciprofloxacin. The antibiotic andcompound combination therapy are preferably administered simultaneouslyor within a period of time sufficient to inhibit the growth of thebiofilm.

In any of the foregoing treatments, the compound and antibiotic includedin the combination therapy are preferably administered to the patient aspart of a pharmaceutical composition that also includes apharmaceutically acceptable carrier. Preferred modes of administrationinclude intramuscular, intravenous, inhalation, and oral administration,or a combination thereof.

The antibiofilm combinations of the invention can also be part of apharmaceutical kit. Preferably, the first compound (e.g., a compoundidentified as modulating expression, at the transcriptional,post-transcriptional, translational, or post-translational levels, of apolynucleotide or polypeptide having at least 50% identity to any one ofthe polynucleotide or polypeptide sequences described herein (or that issubstantially identical to any one of the polynucleotides orpolypeptides described herein)) and the second compound, an antibiotic,are formulated together or separately and in individual dosage amounts.

Combination therapy may be provided wherever antibiotic treatment isperformed: at home, the doctor's office, a clinic, a hospital'soutpatient department, or a hospital. Treatment generally begins at ahospital so that the doctor can observe the therapy's effects closelyand make any adjustments that are needed. The duration of thecombination therapy depends on the kind of biofilm being treated, theage and condition of the patient, the stage and type of the patient'sbiofilm infection, and how the patient's body responds to the treatment.Drug administration may be performed at different intervals (e.g.,daily, weekly, or monthly) and the administration of each agent can bedetermined individually. Combination therapy may be given in on-and-offcycles that include rest periods so that the patient's body has a chanceto build healthy new cells and regain its strength.

By “isolated polynucleotide” is meant a nucleic acid (e.g., a DNA) thatis free of the genes which, in the naturally-occurring genome of theorganism from which the nucleic acid molecule of the invention isderived, flank the gene. The term therefore includes, for example, arecombinant DNA that is incorporated into a vector; into an autonomouslyreplicating plasmid or virus; or into the genomic DNA of a prokaryote oreukaryote; or that exists as a separate molecule (for example, a cDNA ora genomic or cDNA fragment produced by PCR or restriction endonucleasedigestion) independent of other sequences. In addition, the termincludes an RNA molecule which is transcribed from a DNA molecule, aswell as a recombinant DNA which is part of a hybrid gene encodingadditional polypeptide sequence.

By “polypeptide” is meant any chain of amino acids, regardless of lengthor post-translational modification (for example, glycosylation orphosphorylation).

By an “isolated polypeptide” is meant a polypeptide of the inventionthat has been separated from components which naturally accompany it.Typically, the polypeptide is isolated when it is at least 60%, byweight, free from the proteins and naturally-occurring organic moleculeswith which it is naturally associated. Preferably, the preparation is atleast 75%, more preferably at least 90%, and most preferably at least99%, by weight, a polypeptide of the invention. An isolated polypeptideof the invention may be obtained, for example, by extraction from anatural source (for example, a pathogen); by expression of a recombinantnucleic acid encoding such a polypeptide; or by chemically synthesizingthe protein. Purity can be measured by any appropriate method, forexample, column chromatography, polyacrylamide gel electrophoresis, orby HPLC analysis.

By “substantially identical” is meant a polypeptide or nucleic acidmolecule (e.g., a polynucleotide) exhibiting at least 50% identity to areference amino acid sequence (for example, any one of the amino acidsequences described herein) or nucleic acid sequence (for example, anyone of the nucleic acid sequences described herein). Preferably, such asequence is at least 60%, more preferably 80%, and most preferably 90%or even 95% identical at the amino acid level or nucleic acid to thesequence used for comparison.

Sequence identity is typically measured using sequence analysis software(for example, Sequence Analysis Software Package of the GeneticsComputer Group, University of Wisconsin Biotechnology Center, 1710University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, orPILEUP/PRETTYBOX programs). Such software matches identical or similarsequences by assigning degrees of homology to various substitutions,deletions, and/or other modifications. Conservative substitutionstypically include substitutions within the following groups: glycine,alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid,asparagine, glutamine; serine, threonine; lysine, arginine; andphenylalanine, tyrosine. In an exemplary approach to determining thedegree of identity, a BLAST program may be used, with a probabilityscore between e⁻³ and e⁻¹⁰⁰ indicating a closely related sequence.

By “transformed cell” is meant a cell into which (or into an ancestor ofwhich) has been introduced, by means of recombinant DNA techniques, apolynucleotide molecule encoding (as used herein) a polypeptide of theinvention.

By “positioned for expression” is meant that the polynucleotide of theinvention (e.g., a DNA molecule) is positioned adjacent to a DNAsequence which directs transcription and translation of the sequence(i.e., facilitates the production of, for example, a recombinantpolypeptide of the invention, or an RNA molecule).

By “purified antibody” is meant an antibody which is at least 60%, byweight, free from proteins and naturally-occurring organic moleculeswith which it is naturally associated. Preferably, the preparation is atleast 75%, more preferably 90%, and most preferably at least 99%, byweight, antibody. A purified antibody of the invention may be obtained,for example, by affinity chromatography using a recombinantly-producedpolypeptide of the invention and standard techniques.

By “specifically binds” is meant a compound or antibody which recognizesand binds a polypeptide of the invention but which does notsubstantially recognize and bind other molecules in a sample, forexample, a biological sample, which naturally includes a polypeptide ofthe invention.

By “derived from” is meant isolated from or having the sequence of anaturally-occurring sequence (e.g., a cDNA, genomic DNA, synthetic, orcombination thereof).

By “inhibiting biofilm formation” is meant the ability of a candidatecompound to decrease the development or progression of biofilmformation. Preferably, such inhibition decreases biofilm formation by atleast 1% to 5%, more preferably by at least 10%, 15%, 20%, or 25%, andmost preferably by at least 30% to 50%, as compared to biofilm formationin the absence of the candidate compound in any appropriatepathogenicity assay (for example, those assays described herein). In oneparticular example, inhibition is measured by continuous cultureconditions of a microbe exposed to a candidate compound or extract, adecrease in the level of biofilm formation relative to the level ofbiofilm formation of the microbe not exposed to the compound indicatingcompound-mediated inhibition of biofilm formation.

By “biofilm regulator polynucleotide” is meant a polynucleotide encodinga cellular component (e.g., PvrR) that modulates phenotypic switching,such as a phenotypic switch that occurs during biofilm formation,disintegration, or both.

By “phenotypic switching” is meant the reversible alteration of one ormore phenotypic characteristics. Such an alteration typically occurs,for example, when a wild-type microbe develops into anantibiotic-resistant phenotypic variant or when an antibiotic-resistantphenotypic variant develops into a wild-type microbe.

By “immunological assay” is meant an assay that relies on animmunological reaction, for example, antibody binding to an antigen.Examples of immunological assays include ELISAs, Western blots,immunoprecipitations, and other assays known to the skilled artisan.

By a “two-component regulatory system” is meant a regulatory system thatincludes at least two components such as a sensor that senses anenvironmental signal and a response regulator that modulates one or moreeffectors.

By “aggregation” is meant a collection of two or more individualmicroorganisms into a mass or clump, such that the individuals form anaggregated microbial unit. Aggregation can be measured using assaysprovided herein. Examplary assays include visual inspection, measuringattachment to a surface, or by assaying for biofilm formation usingmethods known to the skilled artisan.

By “pathogenicity” is meant the ability of a microorganism to causedisease. A microorganism that forms a biofilm, has increased antibioticresistance, or displays phenotypic variation is more pathogenic than awild-type microorganism in that it is less susceptibile to conventionalantibiotic treatment.

The invention provides a number of targets that are useful for thedevelopment of drugs that specifically block the biofilm formation of amicrobe. In addition, the methods of the invention provide a facilemeans to identify compounds that are safe for use in eukaryotic hostorganisms (i.e., compounds which do not adversely affect the normaldevelopment and physiology of the organism), and efficacious againstpathogenic microbes (i.e., by suppressing the virulence of a pathogen).In addition, the methods of the invention provide a route for analyzingvirtually any number of compounds for an anti-virulence effect withhigh-volume throughput, high sensitivity, and low complexity. Themethods are also relatively inexpensive to perform and enable theanalysis of small quantities of active substances found in eitherpurified or crude extract form.

Other features and advantages of the invention will be apparent from thedetailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the reversion of PA14 rough small colony variants (RSCV)to the wild-type phenotype as observed at the edges of the colonies(arrow) after 2-3 days incubation on antibiotic free LB agar at roomtemperature.

FIG. 1B shows a confocal scanning laser microscopic analysis ofbacterial aggregates (arrows) formed by wild-type PA14 and PA14 RSCVexpressing green fluorescent protein (GFP) after overnight growth inliquid broth. Scale bar, 25 μm.

FIG. 1C shows the attachment of wild-type PA14 and antibiotic resistantvariants to polyvinylchloride plastic (PVC) after 6 hours of growth.

FIG. 1D shows a confocal laser scanning microscope analysis of biofilmformed by wild-type PA14 and PA14 RSCV expressing GFP in flow-chambersunder continuous culture conditions. Scale bar, 50 μm.

FIG. 1E shows PA14 and PA14 RSCV biofilm resistance to tobramycin asdetermined by measuring viable biomass on 45 hour-old establishedbiofilms before (filled bars) and after (open bars) 36-hour tobramycin(200 μg/ml) treatment.

FIG. 2A shows the effect of different environmental stimuli on the rateof appearance of antibiotic resistant variants. This was determined bygrowing the cultures of wild-type PA14 under the specified conditions onmedia containing 200 μg/ml kanamycin.

FIG. 2B shows the minimal inhibitory concentrations of kanamycin forstrain PA14 using the different conditions specified.

FIG. 3A shows the reversion of PA14 RSCV present in sputum samples of acystic fibrosis patient (designated “CF 5”) as observed on the edges ofthe variant colonies (arrow) after prolonged incubation onantibiotic-free medium at room temperature.

FIG. 3B shows the increased attachment to PVC plastic of antibioticresistant variants SCV 42 and SCV 43 obtained after plating CF isolatesCF 42 and CF 43 on tobramycin (10 μg/ml).

FIG. 4A shows the attachment to PVC plastic of PA14, antibioticresistant variants, and PA14 RSCV carrying pEd202 (PA14 RSCV/pED202) orpUCP19 (PA14 RSCV/pUCP19) after 4 hours of growth was quantitated.

FIG. 4B shows the predicted amino acid sequence alignment of PvrR withthe sequences that correspond to VieA from V. Cholerae and the P.aeruginosa PAO1 putative response regulator PA3947 (PAO1 RR). Numbersabove the scale indicate number of amino acids. Lower panel containsdomain family numbers according to ProDom nomenclature.

FIG. 4C shows that the pvrR gene is flanked by two open reading frameregions (ORFs), designated ORF1 and ORF3, with the same transcriptionalorientation. Start codons within ORFs were assigned based on visualinspection for appropriately spaced ribosome-binding sequences.

FIG. 4D shows the number of variants resistant to kanamycin (200 μg/ml).This was evaluated after plating overnight cultures of PA14 and PA14overexpressing PvrR (PA14/pED202).

FIG. 4E shows the attachment to PVC plastic of PA14 and PA14overexpressing PvrR (PA14/pED202) after 12 hours of growth, quantitatedas described herein.

FIG. 4F shows the number of antibiotic resistant variants for PA14 andthe pvrR mutant (ΔpvrR) as determined by plating overnight cultures onLB agar containing kanamycin (200 μg/ml).

FIG. 5A shows the nucleic acid sequence of pvrR (SEQ ID NO:1).

FIG. 5B shows the nucleic acid sequence of an ORF1 polynucleotide (SEQID NO:3). This polynucleotide sequence begins at nucleotide 1504 andends at nucleotide 2919 of SEQ ID NO: 7 as shown in FIG. 5G.

FIG. 5C shows the nucleic acid sequence of an ORF3 polynucleotide (SEQID NO:5). This polynucleotide sequence begins at nucleotide 4385 andends at nucleotide 6379 of SEQ ID NO:7 as shown in FIG. 5G.

FIG. 5D shows the deduced amino acid sequence of PvrR (SEQ ID NO:2).

FIG. 5E shows the deduced amino acid sequence of a polypeptide (SEQ IDNO:4) encoded by the polynucleotide shown in FIG. 5B.

FIG. 5F shows the deduced amino acid sequence of a polypeptide (SEQ IDNO:6) encoded by the polynucleotide shown in FIG. 5C.

FIG. 5G shows the nucleic acid sequence (SEQ ID NO:7) that includes thepvrR gene (SEQ ID NO:1), and the ORF1 (SEQ ID NOS:3 and 8-18) and ORF3(SEQ ID NOS:5 and 30-34) regions. The start and stop codons for theidentified open reading frames are highlighted.

FIGS. 6A-6K show the nucleotide sequences of several open reading framesidentified in the ORF1 region (SEQ ID NO:8 begins at nucleotide 124 andends at nucleotide 2919; SEQ ID NO:9 begins at nucleotide 199 and endsat nucleotide 2919; SEQ ID NO:10 begins at nucleotide 217 and ends atnucleotide 2919; SEQ ID NO:11 begins at nucleotide 256 and ends atnucleotide 2919; SEQ ID NO:12 begins at nucleotide 295 and ends atnucleotide 2919; SEQ ID NO:13 begins at nucleotide 307 and ends atnucleotide 2919; SEQ ID NO:14 begins at nucleotide 511 and ends atnucleotide 2919; SEQ ID NO:15 begins at nucleotide 760 and ends atnucleotide 2919; SEQ ID NO:16 begins at nucleotide 790 and ends atnucleotide 2919; SEQ ID NO:17 begins at nucleotide 919 and ends atnucleotide 2919; and SEQ ID NO18 begins at nucleotide 1429 and ends atnucleotide 2919).

FIGS. 6L-6V show the deduced amino acid sequences of the polypeptides(SEQ ID NOS:19-29) identified in FIGS. 6A-6K above.

FIGS. 7A-7E show the nucleotide sequence of several open reading framesidentified in the ORF3 region (SEQ ID NO:30 begins at nucleotide 4388and ends at nucleotide 6379; SEQ ID NO:31 begins at nucleotide 4550 andends at nucleotide 6379; SEQ ID NO:32 begins at nucleotide 4572 and endsat nucleotide 6379; SEQ ID NO:33 begins at nucleotide 4880 and ends atnucleotide 6379; and SEQ ID NO:34 begins at nucleotide 5258 and ends atnucleotide 6379).

FIGS. 7F-7J show the deduced amino acid sequences of the polypeptides(SEQ ID NOS:35-39) identified in FIGS. 7A-7E above.

DETAILED DESCRIPTION

Overview

Pseudomonas aeruginosa is the most important pathogen in the lungs ofcystic fibrosis (CF) patients. Colonization of the CF lung by P.aeruginosa persists despite the use of long-term antibiotic therapy,since antibiotic treatment rarely results in eradication of theinfection. Reports have suggested a direct link between resistance toantimicrobial compounds and the ability of P. aeruginosa to form biofilmin CF lungs. Other hypotheses explain P. aeruginosa antibioticresistance by postulating that factors within the CF respiratory tractselect for phenotypic variants suited to survive antimicrobialtreatment. As is discussed below, we have determined that a clinicalisolate of P. aeruginosa, strain PA14, was capable of growing underinhibitory concentrations of the antibiotic kanamycin (up to 40 timesthe susceptibility level of the strain) when bacteria had undergonephenotypic variation. The antibiotic resistant variant colonies obtainedfrom kanamycin plates were smaller in size and had a different colonymorphology compared to the wild-type. Analysis of the phenotype of PA14RSCV indicated that these variants exhibited increased aggregation andattachment to glass tubes and polyvinylchloride plastic (PVC) as aresult of enhanced surface hydrophobicity. Consistent with theseobservations, several PA14 RSCV clones were hyperpiliated when analysedby transmission electron microscopy. Moreover, examination of biofilmscultivated in flow chamber cells showed that PA14 RSCV formed morebiofilm and faster than the wild-type strain. The biofilm formed by PA14RSCV also showed increased resistance to tobramycin relative towild-type PA14 biofilm. Similar results were obtained for several CFisolates using different antibiotics (including tobramycin), suggestingthat nonspecific antibiotic resistance acquired through phenotypicvariation is a common mechanism in P. aeruginosa. Moreover, analysis ofsputum samples taken from CF patients revealed that antibiotic treatmentselects for antibiotic resistant variants. The frequency with whichantibiotic resistant variants appeared was also affected byenvironmental stimuli. Environmental stimuli such as salt concentration,temperature, and bacterial media altered the frequency of appearance ofresistant variants.

To identify components involved in the regulation of antibioticresistance mediated by phenotypic variation, a library of PA14chromosomal DNA was transferred into PA14 RSCV and screened for coloniesdisplaying wild-type colony size and morphology. This led to theidentification of a clone, pED202, that restored the colony, theautoagglutination, and attachment phenotypes of PA14 RSCV variants towild-type. pED202 contained a single gene (designated pvrR for phenotypevariant regulator) that showed sequence similarities to responseregulator elements of the two-component regulatory system found inVibrio cholerae response regulator VieA, and in P. aeruginosa strainPAO1 (ORF PA3947).

Consistent with the putative role of PvrR in the regulation ofphenotypic switching, overexpression of PvrR from pED202 in wild-typePA14 resulted in reduced attachment to PVC plastic. Moreover,examination of the frequency of resistant variants obtained fromkanamycin plates showed a reduction in the number of colonies resistantto antibiotic obtained from the PvrR overexpressing strain. An in-framedeletion of pvrR (ΔpvrR) constructed in PA14 increased frequency ofappearance of resistant variants on kanamycin plates with respect to thewild-type, confirming the involvement of pvrR in the regulation ofphenotypic switching. These results suggested that PvrR might be actingupstream of the switch, since inactivation of pvrR by mutation did notresult in conversion to the variant type.

Below we describe the cloning and characterization of PvrR, a regulatorof biofilm-mediated antibiotic resistance and a target for compoundsuseful in antibacterial therapy, along with antibiotics, for thetreatment of chronic infections and biofilm control in medical andindustrial settings. In addition, we describe the identification of openreading frame regions, designated ORF1 and ORF3, that flank the pvrRgene. The following examples are for the purposes of illustrating theinvention, and should not be construed as limiting.

Appearance of Rough Small Colony Variants with Increased AntibioticResistance

When cultured under high concentrations of antibiotic, Pseudomonasaeruginosa PA14 was found to shift its development to a rough smallcolony phenotype, leading to the production of antibiotic resistantcolonies. To induce such phenotypic variants, an overnight culture of P.aeruginosa strain PA14 (UCBPP-PA14) was inoculated onto Luria-Bertani(LB) containing 200 μg/ml of kanamycin, incubated at 37° C. for 48hours, at which time, antibiotic resistant rough small variants wereisolated. Antibiotic resistant colonies arose at a frequency of10⁻⁶-10⁻⁷. The colonies identified on these plates were one-tenth thesize of wild type and exhibited a rough phenotype compared to the smoothcolony type of wild-type PA14. One class of kanamycin resistant variants(approximately 30%) exhibited a rough phenotype compared to the smoothcolony type of wild-type PA14. When incubated for three to five days inLB media without antibiotic at room temperature, the rough phenotypereverted to the wild-type phenotype (FIG. 1A), indicating that thephenotypic changes were transient, and not due to mutation. In additionto being resistant to kanamycin, (up to 40 times the susceptibilitylevel of the wild-type), 8 individual PA14 RSCV colonies tested werealso resistant to amikacin (30 μg/ml), carbenicillin (300 μg/ml),gentamicin (30 μg/ml), tobramycin (10 μg/ml), and tetracycline (150μg/ml). Consistent with this latter result, antibiotic resistantvariants were also obtained at frequencies of about 10⁻⁷ by platingovernight cultures of PA14 on media containing similar concentrations ofthe antibiotics mentioned above. Although RSCV colonies were smallerthan wild-type, their small colony size was not a consequence of slowgrowth since the generation time of RSCV in liquid medium was notsignificantly different from that of the wild-type, even in LBcontaining 200 μg/ml kanamycin.

Phenotypic Changes Associated with Appearance of Resistance

To establish a connection between the phenotypic switch from wild-typeto small variant colony and the emergence of antibiotic resistance,comparative attachment, agglutination, and biofilm formation studies ofwild-type PA14 and PA14 RSCV were conducted.

The results of these experiments showed that PA14 RSCV formed visiblebacterial aggregates when overnight liquid cultures were left withoutshaking at room temperature (FIG. 1B). Moreover, abundant bacterialaggregates formed when liquid cultures were grown with gentle agitation,indicating that PA14 RSCV had increased cell-cell attachment compared tothe wild-type phenotype.

In addition to the autoagglutination phenotype, PA14 RSCV developed avisible biofilm on the walls of glass tubes after overnight incubationin liquid culture. Wild-type PA14 failed to form a similar biofilm underthese conditions. These results indicated that cell-surfaceinteractions, as well as cell-cell interactions were increased in thevariant. Consistent with this observation, PA14 RSCV were found to haveincreased attachment to PVC plastic (FIG. 1C) in assays conducted in96-well microtiter plates. When reversion was induced in PA14 RSCV, thereverted bacteria showed wild-type levels of both agglutination andattachment to glass and PVC plastic.

To quantitatively assess differences between the strains, standardbacterial attachment assays were performed in 96-well polyvinylchloride(PVC) plastic plates according to the methods described by O'Toole etal. (Mol. Microbiol. 30: 295, 1998). Overnight cultures of PA 14 and PA14 RSCV were diluted to an OD₆₀₀ of 0.1 in fresh minimal M63 saltssupplemented with glucose (0.3%), MgSO₄ (1 mM), and casamino acids (CAA,0.5%). Aliquots of 100 μl were next dispensed into the wells of PVCplastic microtiter plates and incubated for 6 hours at 37° C. Theattachment of bacteria to the walls of the microtiter well was thendetected by staining with 1% crystal violet dissolved in water. Dye notassociated with bacteria was removed by thorough rinsing with water.Bacteria-associated dye was solubilized using 95% ethanol and absorbancewas determined at OD₅₅₀.

In addition, since the ability of bacteria to attach to each other andto surfaces depends in part on the interaction of hydrophobic domains(Drumm et al., J. Clin. Invest. 84:1588, 1989), the hydrophobic surfaceproperties of the wild-type and PA14 RSCV were determined using astandard salt aggregation test (Sherman et al., Infect. Immun. 49:797,1985). 5×10⁸ bacteria per ml in 0.025 ml were mixed on a microscopeslide with an equal volume of ammonium sulfate in 0.002 M sodiumphosphate, pH 6.8. The ammonium sulfate concentrations varied from0.0625 M to 4.0 M, and the presence of salt-induced bacterialaggregation was monitored for 2 minutes at room temperature byphase-contrast microscopy. Agglutination in salt concentrations of lessthan 0.1 M is taken as an indication of the presence of a hydrophobicbacterial surface. Hydrophilic surfaces were demonstrated by theagglutination of bacteria only in high salt concentrations (2.0 to 4.0M).

The data obtained from the salt aggregation tests showed that PA14 RSCVwere agglutinated at a lower salt concentration (0.125 M) compared tothe wild-type PA14 (0.5 M), suggesting that PA14 RSCV has a higherdegree of surface hydrophobicity than the wild-type. Therefore, the dataindicated that a change in the hydrophobic properties of the surface ofthe bacteria was partially responsible for the general increase insurface attachment of the PA14 RSCV phenotypic variant. To furtherdemonstrate the role of hydrophobicity in surface attachment, PA14 RSCVwere cultured in the presence of tetramethyl urea (TMU), a hydrophobicbond-breaking agent, at a concentration of 200 mM. Addition of TMU tothe culture media was found to reduce the attachment of the phenotypicvariant PA14 RSCV to wild-type levels, confirming the hydrophobic natureof the bacterial surface. TMU, at the concentration used in theseassays, did not affect cell viability.

Transmission electron microscopic analysis of several PA14 RSCV clonesrevealed that they were hyperpiliated, which is consistent with theincreased hydrophobicity and agglutination phenotypes. However, thevarious phenotypes of PA14 RSCV were not simply a consequence ofhyperpiliation since a hyperpiliated mutant of P. aeruginosa PA14, pilU,exhibited only marginally enhanced hydrophobicity and attachment to PVCplastic and did not exhibit enhanced resistance to antibiotics (data notshown). These results are consistent with previous reports whichindicated that phenotypic variation in Gram-negative bacteria involvechanges in expression of a number of surface structures, outer membraneproteins, and lipopolysaccharides resulting in altered aggregation andcolony morphology. Several PA14 RSCV clones were tested in theexperiments described above and all exhibited similar phenotypes. Asingle PA14 RSCV clone was therefore chosen for further analysis.

To determine whether the antibiotic resistant phenotype of PA14 RSCV isassociated with altered biofilm formation, PA14 RSCV was cultured underbiofilm-forming conditions as follows. For biofilm characterization,PA14 RSCV biofilms were cultivated under continuous culture conditionsin flow-chambers with channel dimensions of 12 by 52 by 2 mm. Flow mediaconsisted of M63 supplemented with 0.5% casamino acids and 0.3% glucose.For measurement of biofilm resistance, bacteria were cultivated inflow-chambers with channel dimensions of 1 by 40 by 4 mm (Stovall Inc.,Greensboro, N.C.). In this case, flow media consisted of FAB medium (0.1mM CaCl₂, 0.01 mM Fe-EDTA, 0.15 mM NH₄SO₄, 0.33 mM Na₂HPO₄, 0.2 mMKH₂PO₄ and 1 mM MgCl₂) supplemented with casamino acids (0.5%) andsodium citrate (10 mM). Flow-cells in both cases were inoculated with100-fold dilutions of overnight cultures of PA14 and PA14 RSCV carryingthe green fluorescent protein (GFP) in plasmid SMC21, a derivative ofpSMC2 (Bloemberg et al., Appl. Environ. Microbiol. 63: 4543-4551, 1997).After inoculation, the medium flow was stopped for 1 hour. Medium flowwas then resumed at a rate of 0.2 ml/min using a peristaltic pump(IsmaTec, Zurich, Switzerland), and the flow-cell system was incubatedat 37° C. Analysis of biofilm spatial structures was performed usingconfocal scanning laser microscopy (CSLM) using a Leica TCS SP system(Leica Lasertechnik, GmgH, Heidelberg, Germany). Image analysis ofantibiotic-treated biofilms was done in structures contained withinserial section stacks of images delimited by freehand drawing. Pixelintensities unique to GFP-labeled bacteria and surrounding biofilm wereestablished by the threshold limit technique. The volume (in μm³) ofindividual biofilm structures was determined from serial sections usingImageSpace software (Molecular Dynamics, Sunnyvale, Calif.).

The results from these studies showed that the PA14 RSCV phenotypicvariant formed not only more biofilm than the wild-type strain, but alsoformed biofilm faster (RSCV microcolonies appeared 4-5 hours earlierthan wild-type). Moreover, PA14 RSCV and wild-type PA14 displayedsignificantly different patterns of biofilm development. Wild-type PA14initially formed regularly-spaced, flat, circular, microcolonies thateventually developed into ball-shaped microcolonies. In contrast, PA14RSCV formed irregularly shaped three-dimensional structures that weredensely packed with bacteria, without the typical microcolony morphology(FIG. 1D). Finally, the biofilm structures formed by PA14 RSCV werelarger in size than the wild-type microcolonies, and biofilms from PA14RSCV contained more biomass than the wild-type.

To determine whether PA14 and PA14 RSCV biofilms exhibited antibioticresistance that paralleled the resistance observed on plates containingantibiotic, established PA14 and PA14 RSCV biofilms grown in flowchambers were exposed to a continuous flow of tobramycin (200 μg/ml).Viable biomass was measured by CSLM analysis of GFP-tagged PA14 and PA14RSCV cells using GFP expression as a viability marker as describedpreviously (FIG. 1E). Consistent with the results obtained in plates,the biofilm formed by PA14 RSCV was more resistant to tobramycintreatment than the wild-type PA14 biofilm.

Phenotypic variation is a common phenomenon in Gram-negative bacteriathat often involves environmentally regulated changes in observablephenotypes produced by modifications in surface components. The effectthat different environmental stimuli had on the appearance ofkanamycin-resistant phenotypic variants was examined. Bacteria weregrown in LB broth, or in supplemented LB with appropriate antibiotics atthe indicated temperature with aeration. As shown in FIG. 2A, a 40-foldincrease in the frequency of appearance of resistant variants (not justPA14 RSCV) was observed on LB media supplemented with 85 mM NaCl ascompared to the same medium without NaCl. Moreover, the frequency ofvariants increased 200-fold when plates were incubated at 25° C.compared to 37° C. (FIG. 2A). Finally, a dramatic 10⁶-fold increase wasobtained on minimal M63 salts as compared to LB medium (FIG. 2A).Minimal salt media consisted of M63 supplemented with 0.3% glucose, 1 mMMgSO₄, and 0.5% casamino acids. Importantly, there was a correlationbetween the frequency of appearance of kanamycin resistant variants onplates and minimal inhibitory concentrations (MICs) of kanamycin inliquid culture for the wild-type PA14 using the culture conditionsdescribed above (FIG. 2B). For example, the high frequency of resistantvariants obtained on M63 correlated with the relatively highconcentration of kanamycin (475 μg/ml) required to inhibit the growth ofPA14 in M63 liquid medium (FIGS. 2A and 2B). These data indicated thatthe components involved in the formation of antibiotic resistantvariants are differentially regulated by environmental signals.Moreover, the data indicated that the portion of the population thatbecomes resistant to antibiotics through phenotypic variation waslargely dependent on environmental conditions.

Small Colony Variants in CF Sputum Samples

The presence of phenotypic variants with small colony phenotypes hasbeen reported in cystic fibrosis (CF) patients (Haussler et al., Clin.Infect. Dis. 29:621, 1999). Emergence of this and other variantphenotypes in the CF lung has also been linked to prolonged antibiotictreatment (McNamara et al., Int. J. Antimicrob. Agents 14:117, 2000;Kahl et al., J. Infect. Dis. 177:1023, 1998). To investigate whetherantibiotic treatment in P. aeruginosa CF infections results in selectionfor resistant variants, we looked for the presence of small colonyvariants in CF sputum samples.

Five CF sputum samples from the Clinical Microbiology Laboratory atMassachusetts General Hospital were suspended in 5 ml of 10 mM MgSO₄.Serial dilutions of the samples were then plated onto cetrimide agarplates with and without antibiotics. The plates were screened for thepresence of P. aeruginosa after 24 and 48 hours of incubation at 37° C.The identity of the colonies was later confirmed by probing colony liftswith the exotoxin A gene from P. aeruginosa. To this end, theEcoRI-HindIII fragment of plasmid pRGI containing the exoA gene(Samadpour et al., J. Clin. Microbiol. 26:2319-23, 1988) was gelisolated and labeled using a random priming kit (Boehringer, Mannheim,Indianapolis, Ind.). Colonies were transferred to nylon membranes andhybridizations were performed according to the manufacturer'srecommendations (NEN Research Products, Boston, Mass.). Identificationof colonies carrying the exoA gene was then performed using aPhosphorimager (Amersham Pharmacia Biotech Inc., Piscataway, N.J.).

Five sputum samples obtained from five CF patients were evaluated forthe presence of small colony variant bacteria. Two out of five sputumsamples obtained from CF patients (patients 5 and 38) contained 100%rough small colony variants (Table 1) that reverted to a wild-typecolony morphology upon prolonged incubation on antibiotic-free medium(FIG. 3A). Importantly, both samples 5 and 38 corresponded to patientsthat were undergoing antibiotic treatment at the time the samples wereobtained (intravenous (IV) amikacin/ceftazidime for two days and oral(O) levofloxacin/inhaled (I) tobramycin for six weeks respectively Table1). TABLE 1 Sample 5 Sample 38 Sample 41 Sample 42 Sample 43 Antibiotictreatment of CF Amikacin(IV) Tobramycin (I) none none none patientsCeftazidime(IV) Levofloxacin(0) Small Colony variants in 100 100 <0.110.00 <0.12 sputum sample (%) Variants resistant to amikacin 100 100 15 50.2 (%) Variants resistant to gentamicin 100 100 10 6.6 0.5 (%) Variantsresistant to 30 32 0 0 Not done tetracycline (%) Variants resistant to50 100 0.10 0 0.5 tobramycin (%)

Table 1 shows the presence of small colony P. aeruginosa variants insputum samples from five CF patients. The presence of P. aeruginosaantibiotic resistant small colony variants was determined by plating CFsputum samples on cetrimide agar with and without the indicatedantibiotics.

Moreover, there was 29% enrichment in small colony variants in samplestaken on two consecutive days from the patient that was undergoingintravenous antibiotic treatment.

As shown in Table 1, 30-100% of the small colony variants present insamples 5 and 38 were resistant to four different antibiotics (amikacin,gentamicin, tetracycline, and tobramycin) at concentrations equal to orhigher than the minimal bactericidal concentration (MBC) of theirrespective reverted colonies. The proportion of small colony variantspresent in the samples that showed resistance to amikacin, gentamicin,tetracycline, and tobramycin was analyzed by simultaneously plating thesputum samples in cetrimide agar with and without antibiotics. The dataobtained were compared to MBCs of the reverted colonies for theantibiotics in which variants were obtained In vitro susceptibility(MBC) to the different antibiotics used during the assays was determinedby a standard tube dilution procedure described by Bailey and Scott(Diagnostic Microbiology, 313-329, 1974).

Although the other three CF sputum samples (41, 42 and 43) appeared tocontain either a small proportion or no detectable small colony variantswhen plated on antibiotic free media, they did contain a considerablenumber (0.5-15%) of antibiotic resistant variants (Table 1). Thisdiscrepancy was due to the fact that it took the small colony variants36-40 hours to form visible colonies, at which time the fast growingwild-type bacteria present in the sputum samples had overgrown theantibiotic free plates. Resistant variants with small colony phenotypesobtained from plating CF isolates 42 and 43 on media containingtobramycin (a front-line antibiotic used for the treatment of P.aeruginosa infections) exhibited increased attachment to PVC plastic(FIG. 3B).

Identification of the Phenotypic Variation Regulator Gene

Phenotypic variation is a common mechanism in Gram-negative bacteria,and involves changes in observable phenotypes produced by modificationsin surface components such as fimbriae, flagella, outer membraneproteins, and lipopolysaccharides. In the mushroom pathogen P. tolaasii,Greewal et al. (J. Bacteriol. 177:4658, 1995) identified a two-componentregulatory element responsible for the phenotypic switch from smooth torough phenotype that involved changes in colony morphology and motility.Since the phenotype displayed by PA14 RSCV was transient and involvedalterations in surface properties, we hypothesized that a regulatorycomponent was also responsible for the phenotypic switch observed inPA14.

To identify this component, a genomic library of strain PA14 constructedin the cosmid vector pJSR1 (Rahme et al., Science 268:1899, 1995) wasmobilized in masse into PA14 RSCV by triparental mating using helperstrain pRK2013 (Figurski et al., Proc. Natl. Acad. Sci. USA 76:1648,1979). The resulting transconjugants were screened visually for coloniesshowing wild-type size and morphology (smooth colony phenotype).Individual transconjugants that showed wild-type characteristics wereused to isolate the corresponding cosmids which were then reintroducedinto PA14 RSCV to confirm the reversion of the phenotype. Moreover,cosmid DNA from the transconjugants was digested to completion with therestriction enzymes EcoRI, PstI, and HindIII and separated byelectrophoresis on a 0.7% agarose gel.

A total of 2,500 transconjugants were screened for colonies displayingwild-type PA14 colony size and morphology. Two transconjugants thatshowed wild-type phenotypes were isolated, indicating that the insertscontained in the cosmids were able to induce reversion from small colonyvariant to wild-type phenotype. Two cosmid clones were isolated andreintroduced in PA14 RSCV to test for restoration of wild-typephenotype, and both clones were found to be capable of greatly enhancingthe rate of PA14 RSCV reversion to the wild-type phenotype. Restrictiondigest profiles obtained with EcoRI, PstI, and HindIII restrictionenzymes showed the presence of a cosmid with the same insert in bothcases, which was designated pED20. Although the PA14 RSCV phenotype wasnormally very stable in liquid culture (i.e., no wild-type revertantsobserved when an overnight culture was plated on LB agar), the majorityof the cells in a PA14 RSCV culture carrying pED20 formed wild-typecolonies after overnight incubation.

Cosmid pED20 was then subcloned into the pUCP19 plasmid vector using aPstI restriction digest. The clones obtained after transformation in E.coli were used to isolate plasmid DNA that was subsequently introducedinto PA14 RSCV by electroporation. The resulting clones were screenedvisually for colonies showing wild-type size and morphology. Subcloningof pED20 produced pED202, which contained a 3.5-kb fragment, thatrestored the colony phenotype of PA14 RSCV variant to wild-type. ClonepED202 restored attachment phenotypes (FIG. 4A), as well as the colonymorphology and autoagglutination phenotypes of PA14 RSCV variants towild-type. The vector alone did not have any effect on the phenotypesanalyzed.

DNA sequencing and sequence analysis of the pED202 insert was thenperformed. The DNA fragments used for sequencing were PCR amplifiedinitially using primers M13 and M13 reverse from the pUCP19 plasmid.Primers were later synthesized based on the sequencing data obtained.Sequencing data were analyzed using the DNAStar software (DNASTAR Inc.,Madison, Wis.) to predict the open reading frames present in the pED2023.5 kb insert. Sequence information was also compared with the sequencedatabases at the National Center for Biotechnology Information as wellas to the P. aeruginosa PAO1 sequence generated by the P. aeruginosagenome project (Cystic Fibrosis Foundation and PathoGenesisCorporation).

Analysis of the sequencing data obtained from clone pED202 showed thatthe clone contained only one intact open reading frame. The nucleotideand predicted amino acid sequences of the ORF (designated pvrR forphenotype variant regulator) contained in clone pED202 were compared tothe GenBank databases, and showed sequence similarities to responseregulator elements of the two-component regulatory system. The searchrevealed 30% identity and 45% similarity in a 376 amino acid overlap tothe Vibrio cholerae response regulator VieA, which is induced duringintestinal infection in mouse. In addition, the ORF on pED202 showed 29%identity and 45% similarity to a probable two-component responseregulator identified in P. aeruginosa strain PAO1 (ORF PA3947).Interestingly, the region of the PA14 genome containing pvrR is notpresent in the fully sequence P. aeruginosa strain PAO1.

A homology search against domain sequences in the ProDom database(ProDom web site; http://prodes.Toulouse.inra.fr/prodom) identified 4regions with high-scoring segment pairs in PvrR (FIG. 4B). All 4 domainsare also present in VieA and the PA01 putative response regulator (FIG.4B). Moreover, these 4 domains exhibit high levels of amino acidsequence similarity (30%-60%; FIG. 4B). Sequence analysis of the regionslocated upstream and downstream of pvrR revealed the presence of twoadditional ORFs (designated ORF1 and ORF3 respectively; FIG. 4C) withsequence homology to two-component regulatory elements.

The protein encoded by ORF1 has homology to probable sensor/responseregulator hybrids from P. aeruginosa (35% identity and 49% similarity toORF. PA2824), to the sensor protein RcsC (capsular synthesis regulatorcomponent C) from Salmonella enterica subsp. enterica serovar Typhi (30%identity and 51% similarity) and to a two-component sensor regulator(PheN) that modulates phenotypic switching in P. tolaasii, (31% identityand 45% similarity). The protein encoded by ORF3 shows 42% identity and60% similarity to the GacS sensor kinase from P. fluorescens, and 41%identity and 59% similarity to the two-component sensor regulator thatmodulates phenotypic switching in P. tolaasii (PheN).

FIG. 5G shows a nucleic acid sequence (SEQ ID NO:7) includingpolynucleotides identified in the ORF1 region (SEQ ID NOS:3, and 8-18),pvrR (SEQ ID NO:1), polynucleotides identified in the ORF3 region (SEQID NOS:5, and 30-34), and the intergenic regions. The start and stopcodons for each open reading frame are indicated by highlighting. FIGS.5B and 6A-K show the nucleotide sequences of several open reading framesidentified in the ORF1 region. The deduced amino acid sequence of theseopen reading frames are shown in FIGS. 5E (SEQ ID NO:4) and 6L-6V (SEQID NOS:19-29).

Additionally, FIG. 5C shows the nucleic acid sequence (SEQ ID NO:5) ofone of several open reading frames identified in the ORF3 region. Thededuced amino acid sequence of the polypeptide encoded by thisnucleotide sequence is shown in FIG. 5F (SEQ ID NO:6). FIGS. 7A-7E (SEQID NOS:30-34) show the nucleotide sequences of several additional openreading frames identified in the ORF3 region. The deduced amino acidsequence of the polypeptides encoded by these nucleotide sequences areshown in FIGS. 7F-7J.

To determine whether pvrR or a highly similar pvrR homolog was presentin the other P. aeruginosa strains, PCR analysis of 14 P. aeruginosastrains was performed using pvR-specific primers. The specificity of thePCR products obtained was subsequently confirmed by Southern blottingand hybridization with a pvrR-specific probe. Results showed that 7 outof 7 CF isolates, 2 out of 3 clinical isolates and 3 out of 4 standardP. aeruginosa laboratory strains contained the pvrR gene fragment or ahighly similar fragment (data not shown).

PvrR Overexpression

Consistent with the putative role of PvrR in the regulation ofphenotypic switching, overexpression of PvrR from pED202 resulted in a6-fold reduction in the frequency of resistant variants obtained afterplating overnight cultures on kanamycin (200 μg/ml) plates compared towild-type (FIG. 4D). Plasmid pED202, containing the pvrR gene wasintroduced into wild-type PA14 by electoroporation using standardmethods. Frequency of appearance of kanamycin resistant variants andattachment to 96-well PVC plates was assayed as described above.Interestingly, the PvrR overexpressing strain also caused a 2.5-foldreduction in attachment to PVC plastic with respect to the straincarrying the vector alone (FIG. 4E).

PvrR Deletion Analysis

Since PvrR is involved in the regulation of the phenotypic switch fromwild-type to phenotypic variant, a mutation in pvrR would be expected toalter the proportion of resistant variants present in the PA14population. To test this hypothesis, a 914 bp in-frame deletion withinpvrR (denoted “ΔpvrR”) was generated by replacing 2.33 kb of thewild-type sequence of the pvrR gene with a 1.416 kb fragment amplifiedby PCR. The PCR-amplified DNA fragment was subcloned into the XbaI andSmaI restriction sites of the positive selection suicide vector pCVD442to generate pED167. Plasmid pED167 was then used in an allelic exchangeprocedure to introduce the fragment containing the deleted copy of pvrRinto the homologous region of the PA14 chromosome, creating strain ED78.The deletion was confirmed by sequencing a PCR fragment containing pvrR.

This deletion of pvrR (ΔpvrR) in PA14 resulted in an increased frequencyof appearance of resistant variants on kanamycin plates with respect tothe wild-type (FIG. 4F), confirming the involvement of pvrR in theregulation of phenotypic switching. The observation that 100% of thevariants expressing wild-type pvrR reverted to the wild-type phenotypeimplicates PvrR is inducing reversion from variant to wild-typephenotypes. These results indicated that PvrR may be acting upstream ofthe switch, since inactivation of pvrR by mutation was not found toresult in conversion to the variant type.

Isolation of Additional Biofilm Regulator Genes

Based on the nucleotide and amino acid sequences described herein, theisolation and identification of additional coding sequences of genesregulating the formation of microbial biofilm is made possible usingstandard strategies and techniques that are well known in the art. Forexample, any microbe that possesses the ability to form a biofilm canserve as the nucleic acid source for the molecular cloning of such agene, and these sequences are identified as ones encoding a proteinexhibiting structures, properties, or activities associated with biofilmformation, such as the PvrR (FIG. 5D, SEQ ID NO:2), or any of thepolynucleotides identified in the ORF1 (SEQ ID NOS:3 and 8-18) and ORF3(SEQ ID NOS:5 and 30-34) regions.

In one particular example of such an isolation technique, any one of thenucleotide sequences described herein, including pvrR (FIG. 5A, SEQ IDNO:1), ORF1 (FIG. 5B, SEQ ID NO:3), or ORF3 (FIG. 5C, SEQ ID NO:5) maybe used, together with conventional methods of nucleic acidhybridization screening. Such hybridization techniques and screeningprocedures are well known to those skilled in the art and are described,for example, in Benton and Davis (Science 196:180, 1977); Grunstein andHogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al.(Current Protocols in Molecular Biology, Wiley Interscience, New York,2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987,Academic Press, New York); and Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, New York. In oneparticular example, all or part of the pvrR, ORF1, or ORF3 sequences(described herein) may be used as a probe to screen a recombinant DNAlibrary for genes having sequence identity to the pvrR, ORF1, or ORF3genes. Hybridizing sequences are detected by plaque or colonyhybridization according to standard methods.

Alternatively, using all or a portion of the amino acid sequences ofPvrR, ORF1, or ORF3, one may readily design pvrR, ORF1, or ORF3gene-specific oligonucleotide probes, including degenerateoligonucleotide probes (i.e., a mixture of all possible coding sequencesfor a given amino acid sequence). These oligonucleotides may be basedupon the sequence of either DNA strand and any appropriate portion ofthe pvrR, ORF1, or ORF3 sequences. General methods for designing andpreparing such probes are provided, for example, in Ausubel et al.(supra), and Berger and Kimmel, Guide to Molecular Cloning Techniques,1987, Academic Press, New York. These oligonucleotides are useful forpvrR, ORF1, or ORF3 gene isolation, either through their use as probescapable of hybridizing to pvrR, ORF1, or ORF3 complementary sequences oras primers for various amplification techniques, for example, polymerasechain reaction (PCR) cloning strategies. If desired, a combination ofdifferent, detectably-labelled oligonucleotide probes may be used forthe screening of a recombinant DNA library. Such libraries are preparedaccording to methods well known in the art, for example, as described inAusubel et al. (supra), or they may be obtained from commercial sources.

As discussed above, sequence-specific oligonucleotides may also be usedas primers in amplification cloning strategies, for example, using PCR.PCR methods are well known in the art and are described, for example, inPCR Technology, Erlich, ed., Stockton Press, London, 1989; PCRProtocols: A Guide to Methods and Applications, Innis et al., eds.,Academic Press, Inc., New York, 1990; and Ausubel et al. (supra).Primers are optionally designed to allow cloning of the amplifiedproduct into a suitable vector, for example, by including appropriaterestriction sites at the 5′ and 3′ ends of the amplified fragment (asdescribed herein). If desired, nucleotide sequences may be isolatedusing the PCR “RACE” technique, or Rapid Amplification of cDNA Ends(see, e.g., Innis et al. (supra)). By this method, oligonucleotideprimers based on a desired sequence are oriented in the 3′ and 5′directions and are used to generate overlapping PCR fragments. Theseoverlapping 3′- and 5′-end RACE products are combined to produce anintact full-length cDNA. This method is described in Innis et al.(supra); and Frohman et al., Proc. Natl. Acad. Sci. USA 85:8998, 1988.

Partial sequences, e.g., sequence tags, are also useful as hybridizationprobes for identifying full-length sequences, as well as for screeningdatabases for identifying previously unidentified related virulencegenes.

In general, the invention includes any nucleic acid sequence which maybe isolated as described herein or which is readily isolated by homologyscreening or PCR amplification using any of the nucleic acid sequencesdisclosed herein such as those shown in FIGS. 5A, 5C, 5G, 6A-K, or7A-7E.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of polynucleotidesequences encoding PvrR, ORF1, or ORF3, some bearing minimal similarityto the polynucleotide sequences of any known and naturally occurringgene, may be produced. Thus, the invention contemplates each and everypossible variation of polynucleotide sequence that could be made byselecting combinations based on possible codon choices. Thesecombinations are made in accordance with the standard triplet geneticcode as applied to the polynucleotide sequence of naturally-occurringpvrR, ORF1, or ORF3, and all such variations are to be considered asbeing specifically disclosed.

Although nucleotide sequences which encode PvrR, ORF1, ORF3, or theirvariants are preferably capable of hybridizing to the nucleotidesequence of the naturally-occurring pvrR, ORF1, or ORF3 underappropriately selected conditions of stringency, it may be advantageousto produce nucleotide sequences encoding PvrR, ORF1, ORF3, or theirderivatives possessing a substantially different codon usage, e.g.,inclusion of non-naturally occurring codons. Codons may be selected toincrease the rate at which expression of the peptide occurs in aparticular prokaryotic or eukaryotic host in accordance with thefrequency with which particular codons are utilized by the host. Otherreasons for substantially altering the nucleotide sequence encodingPvrR, ORF1, ORF3, and their derivatives without altering the encodedamino acid sequences include the production of RNA transcripts havingmore desirable properties, such as a greater half-life, than transcriptsproduced from the naturally occurring sequence.

The invention also encompasses production of DNA sequences which encodePvrR, ORF1, ORF3, or fragments thereof generated entirely by syntheticchemistry. After production, the synthetic sequence may be inserted intoany of the many available expression vectors and cell systems usingreagents well known in the art. Moreover, synthetic chemistry may beused to introduce mutations into a sequence encoding any one of PvrR,ORF1, ORF3, or any fragment thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed polynucleotide sequences, and, inparticular, to those shown in FIG. 5A, 5B, 5C, 5G, 6A-6K, or 7A-7E andfragments thereof under various conditions of stringency. (See, e.g.,Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A.R. (1987) Methods Enzymol. 152:507) For example, stringent saltconcentration will ordinarily be less than about 750 mM NaCl and 75 mMtrisodium citrate, preferably less than about 500 mM NaCl and 50 mMtrisodium citrate, and most preferably less than about 250 mM NaCl and25 mM trisodium citrate. Low stringency hybridization can be obtained inthe absence of organic solvent, e.g., formamide, while high stringencyhybridization can be obtained in the presence of at least about 35%formamide, and most preferably at least about 50% formamide. Stringenttemperature conditions will ordinarily include temperatures of at leastabout 30° C., more preferably of at least about 37° C., and mostpreferably of at least about 42° C. Varying additional parameters, suchas hybridization time, the concentration of detergent, e.g., sodiumdodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA,are well known to those skilled in the art. Various levels of stringencyare accomplished by combining these various conditions as needed. In apreferred embodiment, hybridization will occur at 30° C. in 750 mM NaCl,75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment,hybridization will occur at 37° C. in 500 mM NaCl, 50 mM trisodiumcitrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA(ssDNA). In a most preferred embodiment, hybridization will occur at 42°C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and200 μg/ml ssDNA. Useful variations on these conditions will be readilyapparent to those skilled in the art.

The washing steps which follow hybridization can also vary instringency. Wash stringency conditions can be defined by saltconcentration and by temperature. As above, wash stringency can beincreased by decreasing salt concentration or by increasing temperature.For example, stringent salt concentration for the wash steps willpreferably be less than about 30 mM NaCl and 3 mM trisodium citrate, andmost preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.Stringent temperature conditions for the wash steps will ordinarilyinclude temperature of at least about 25° C., more preferably of atleast about 42° C., and most preferably of at least about 68° C. In apreferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washsteps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and0.1% SDS. In a most preferred embodiment, wash steps will occur at 68°C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additionalvariations on these conditions will be readily apparent to those skilledin the art.

Methods for DNA sequencing are well known in the art and may be used topractice any of the embodiments of the invention. The resultingsequences are analyzed using a variety of algorithms which are wellknown in the art. (See, e.g., Ausubel, F. M. (1997) Short Protocols inMolecular Biology, John Wiley & Sons, New York N.Y., unit 7.7).

Polypeptide Expression

In general, polypeptides of the invention (e.g., PvrR, ORF1, or ORF3 asshown in FIGS. 5D, 5E, 5F, 6L-6V, or 7F-7J) may be produced bytransformation of a suitable host cell with all or part of apolypeptide-encoding nucleic acid molecule or fragment thereof in asuitable expression vehicle.

Those skilled in the field of molecular biology will understand that anyof a wide variety of expression systems may be used to provide therecombinant protein. The precise host cell used is not critical to theinvention. A polypeptide of the invention may be produced in aprokaryotic host (e.g., E. coli) or in a eukaryotic host (e.g.,Saccharomyces cerevisiae, insect cells, e.g., Sf21 cells, or mammaliancells, e.g., NIH 3T3, HeLa, or preferably COS cells). Such cells areavailable from a wide range of sources (e.g., the American Type CultureCollection, Rockland, Md.; also, see, e.g., Ausubel et al., supra). Themethod of transformation or transfection and the choice of expressionvehicle will depend on the host system selected. Transformation andtransfection methods are described, e.g., in Ausubel et al. (supra);expression vehicles may be chosen from those provided, e.g., in CloningVectors: A Laboratory Manual (P. H. Pouwels et al., 1985, Supp. 1987).

One particular bacterial expression system for polypeptide production isthe E. coli pET expression system (Novagen, Inc., Madison, Wis.).According to this expression system, DNA encoding a polypeptide isinserted into a pET vector in an orientation designed to allowexpression. Since the gene encoding such a polypeptide is under thecontrol of the T7 regulatory signals, expression of the polypeptide isachieved by inducing the expression of T7 RNA polymerase in the hostcell. This is typically achieved using host strains which express T7 RNApolymerase in response to IPTG induction. Once produced, recombinantpolypeptide is then isolated according to standard methods known in theart, for example, those described herein.

Another bacterial expression system for polypeptide production is thepGEX expression system (Pharmacia). This system employs a GST genefusion system which is designed for high-level expression of genes orgene fragments as fusion proteins with rapid purification and recoveryof functional gene products. The protein of interest is fused to thecarboxyl terminus of the glutathione S-transferase protein fromSchistosoma japonicum and is readily purified from bacterial lysates byaffinity chromatography using Glutathione Sepharose 4B. Fusion proteinscan be recovered under mild conditions by elution with glutathione.Cleavage of the glutathione S-transferase domain from the fusion proteinis facilitated by the presence of recognition sites for site-specificproteases upstream of this domain. For example, proteins expressed inpGEX-2T plasmids may be cleaved with thrombin; those expressed inpGEX-3X may be cleaved with factor Xa.

Once the recombinant polypeptide of the invention is expressed, it isisolated, e.g., using affinity chromatography. In one example, anantibody (e.g., produced as described herein) raised against apolypeptide of the invention may be attached to a column and used toisolate the recombinant polypeptide. Lysis and fractionation ofpolypeptide-harboring cells prior to affinity chromatography may beperformed by standard methods (see, e.g., Ausubel et al., supra).

Once isolated, the recombinant protein can, if desired, be furtherpurified, e.g., by high performance liquid chromatography (see, e.g.,Fisher, Laboratory Techniques In Biochemistry And Molecular Biology,eds., Work and Burdon, Elsevier, 1980).

Polypeptides of the invention, particularly short peptide fragments, canalso be produced by chemical synthesis (e.g., by the methods describedin Solid Phase Peptide Synthesis, 2nd ed., 1984 The Pierce Chemical Co.,Rockford, Ill.). Also included in the invention are polypeptides whichare modified in ways which do not abolish their pathogenic activity(assayed, for example as described herein). Such changes may includecertain mutations, deletions, insertions, or post-translationalmodifications, or may involve the inclusion of any of the polypeptidesof the invention as one component of a larger fusion protein.

The invention further includes analogs of any naturally-occurringpolypeptide of the invention. Analogs can differ from thenaturally-occurring the polypeptide of the invention by amino acidsequence differences, by post-translational modifications, or by both.Analogs of the invention will generally exhibit at least 85%, morepreferably 90%, and most preferably 95% or even 99% identity with all orpart of a naturally-occurring amino acid sequence of the invention. Thelength of sequence comparison is at least 15 amino acid residues,preferably at least 25 amino acid residues, and more preferably morethan 35 amino acid residues. Again, in an exemplary approach todetermining the degree of identity, a BLAST program may be used, with aprobability score between e⁻³ and e⁻¹⁰⁰ indicating a closely relatedsequence. Modifications include in vivo and in vitro chemicalderivatization of polypeptides, e.g., acetylation, carboxylation,phosphorylation, or glycosylation; such modifications may occur duringpolypeptide synthesis or processing or following treatment with isolatedmodifying enzymes. Analogs can also differ from the naturally-occurringpolypeptides of the invention by alterations in primary sequence. Theseinclude genetic variants, both natural and induced (for example,resulting from random mutagenesis by irradiation or exposure toethanemethylsulfate or by site-specific mutagenesis as described inSambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual(2d ed.), CSH Press, 1989, or Ausubel et al., supra). Also included arecyclized peptides, molecules, and analogs which contain residues otherthan L-amino acids, e.g., D-amino acids or non-naturally occurring orsynthetic amino acids, e.g., β or γ amino acids.

In addition to full-length polypeptides, the invention also includesfragments of any one of the polypeptides of the invention. As usedherein, the term “fragment,” means at least 5, preferably at least 20contiguous amino acids, preferably at least 30 contiguous amino acids,more preferably at least 50 contiguous amino acids, and most preferablyat least 60 to 80 or more contiguous amino acids. Fragments of theinvention can be generated by methods known to those skilled in the artor may result from normal protein processing (e.g., removal of aminoacids from the nascent polypeptide that are not required for biologicalactivity or removal of amino acids by alternative mRNA splicing oralternative protein processing events). The aforementioned generaltechniques of polypeptide expression and purification can also be usedto produce and isolate useful peptide fragments or analogs (describedherein).

Antibodies

The polypeptides disclosed herein or variants thereof or cellsexpressing them can be used as an immunogen to produce antibodiesimmunospecific for such polypeptides. “Antibodies” as used hereininclude monoclonal and polyclonal antibodies, chimeric, single chain,simianized antibodies and humanized antibodies, as well as Fabfragments, including the products of an Fab immunolglobulin expressionlibrary.

To generate antibodies, a coding sequence for a polypeptide of theinvention may be expressed as a C-terminal fusion with glutathioneS-transferase (GST) (Smith et al., Gene 67:31, 1988). The fusion proteinis purified on glutathione-Sepharose beads, eluted with glutathione,cleaved with thrombin (at the engineered cleavage site), and purified tothe degree necessary for immunization of rabbits. Primary immunizationsare carried out with Freund's complete adjuvant and subsequentimmunizations with Freund's incomplete adjuvant. Antibody titres aremonitored by Western blot and immunoprecipitation analyses using thethrombin-cleaved protein fragment of the GST fusion protein. Immune seraare affinity purified using CNBr-Sepharose-coupled protein. Antiserumspecificity is determined using a panel of unrelated GST proteins.

As an alternate or adjunct immunogen to GST fusion proteins, peptidescorresponding to relatively unique immunogenic regions of a polypeptideof the invention may be generated and coupled to keyhole limpethemocyanin (KLH) through an introduced C-terminal lysine. Antiserum toeach of these peptides is similarly affinity purified on peptidesconjugated to BSA, and specificity tested in ELISA and Western blotsusing peptide conjugates, and by Western blot and immunoprecipitationusing the polypeptide expressed as a GST fusion protein.

Alternatively, monoclonal antibodies which specifically bind any one ofthe polypeptides of the invention are prepared according to standardhybridoma technology (see, e.g., Kohler et al., Nature 256:495, 1975;Kohler et al., Eur. J. Immunol. 6:511, 1976; Kohler et al., Eur. J.Immunol. 6:292, 1976; Hammerling et al., In Monoclonal Antibodies and TCell Hybridomas, Elsevier, N.Y., 1981; Ausubel et al., supra). Onceproduced, monoclonal antibodies are also tested for specific recognitionby Western blot or immunoprecipitation analysis (by the methodsdescribed in Ausubel et al., supra. Antibodies which specificallyrecognize the polypeptide of the invention are considered to be usefulin the invention; such antibodies may be used, e.g., in an immunoassay.Alternatively monoclonal antibodies may be prepared using thepolypeptide of the invention described above and a phage display library(Vaughan et al., Nature Biotech 14:309, 1996).

Preferably, antibodies of the invention are produced using fragments ofthe polypeptides disclosed herein which lie outside generally conservedregions and appear likely to be antigenic, by criteria such as highfrequency of charged residues. In one specific example, such fragmentsare generated by standard techniques of PCR and cloned into the pGEXexpression vector (Ausubel et al., supra). Fusion proteins are expressedin E. coli and purified using a glutathione agarose affinity matrix asdescribed in Ausubel et al. (supra). To attempt to minimize thepotential problems of low affinity or specificity of antisera, two orthree such fusions are generated for each protein, and each fusion isinjected into at least two rabbits. Antisera are raised by injections ina series, preferably including at least three booster injections.

Antibodies against any of the polypeptides described herein may beemployed to treat bacterial infections, for example, those infectionsinvolving biofilm formation. Thus, among others, antibodies against, forexample, polypeptides of PvrR (SEQ ID NO: 2), ORF1 (SEQ ID NO: 4), orORF3 (SEQ ID NO: 6) shown respectively in FIGS. 5D, E, or F may beemployed to treat infections, particularly bacterial infections andespecially chronic infections associated with CF or biofilm formationassociated with indwelling medical devices, conjunctivitis, pneumonia,and bacteremia.

Diagnostics

In another embodiment, antibodies which specifically bind any of thepolypeptides described herein may be used for the diagnosis of bacterialinfection. A variety of protocols for measuring such polypeptides,including ELISAs, RIAs, and FACS, are known in the art and provide abasis for diagnosing bacterial infections.

In another aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding pvrR, ORF1, ORF3, or closely related molecules may be used toidentify nucleic acid sequences which encode its gene product. Thespecificity of the probe, whether it is made from a highly specificregion, e.g., the 5′ regulatory region, or from a less specific region,e.g., a conserved motif, and the stringency of the hybridization oramplification (maximal, high, intermediate, or low), will determinewhether the probe identifies only naturally occurring sequences encodingPvrR, ORF1, or ORF3 allelic variants, or related sequences.

In further embodiments, oligonucleotides or longer fragments derivedfrom any of the polynucleotide sequences described herein may be used astargets in a microarray. The microarray can be used to monitor theexpression level of large numbers of genes simultaneously and toidentify genetic variants, mutations, and polymorphisms. Thisinformation may be used to determine gene function, to understand thegenetic basis of a disorder, to diagnose a disorder, and to develop andmonitor the activities of therapeutic agents. Microarrays may beprepared, used, and analyzed using methods known in the art. (See, e.g.,Brennan et al., U.S. Pat. No. 5,474,796; Schena et al., Proc. Natl.Acad. Sci. 93:10614, 1996; Baldeschweiler et al., PCT applicationWO95/251116, 1995; Shalon, D. et al., PCT application WO95/35505, 1995;Heller et al., Proc. Natl. Acad. Sci. 94:2150, 1997; and Heller et al.,U.S. Pat. No. 5,605,662.)

Screening Assays

As discussed above, we have identified a biofilm regulator gene, pvrR,of P. aeruginosa that mediates biofilm formation and antibioticresistance by a microbe. Based on this discovery, we have developedscreening assays for identifying compounds that enhance or inhibit theaction of a polypeptide or the expression of a nucleic acid sequence ofthe invention. The method of screening may involve high-throughputtechniques.

Any number of methods are available for carrying out such screeningassays. In one working example, candidate compounds are added at varyingconcentrations to the culture medium of pathogenic cells expressing oneof the nucleic acid sequences of the invention. Gene expression is thenmeasured, for example, by standard Northern blot analysis (Ausubel etal., supra) or RT-PCR, using any appropriate fragment prepared from thenucleic acid molecule as a hybridization probe. The level of geneexpression in the presence of the candidate compound is compared to thelevel measured in a control culture medium lacking the candidatemolecule. A compound which promotes an increase in the expression of thepvrR gene or functional equivalent is considered useful in theinvention; such a molecule may be used, for example, as a therapeutic tocombat the pathogenicity of an infectious organism, for example, bydecreasing its ability to form a biofilm and rendering it susceptible toantibiotic treatment.

In another working example, the effect of candidate compounds may bemeasured at the level of polypeptide production using the same generalapproach and standard immunological techniques, such as Western blottingor immunoprecipitation with an antibody specific for a biofilm regulatorpolypeptide, such as PvrR. For example, immunoassays may be used todetect or monitor the expression of at least one of the polypeptides ofthe invention in a microbial organism. Polyclonal or monoclonalantibodies (produced as described above) which are capable of binding tosuch a polypeptide may be used in any standard immunoassay format (e.g.,ELISA, Western blot, or RIA assay) to measure the level of thepolypeptide. A compound which promotes an increase in the expression ofthe polypeptide is considered particularly useful. Again, such amolecule may be used, for example, as a therapeutic to combat thebiofilm formation of an organism as is described above.

In yet another working example, candidate compounds may be screened forthose which specifically bind to and agonize a PvrR polypeptide (apolypeptide having the amino acid sequences shown in FIG. 5D) of theinvention. The efficacy of such a candidate compound is dependent uponits ability to interact with the PvrR polypeptide or functionalequivalent thereof. Such an interaction can be readily assayed using anynumber of standard binding techniques and functional assays (e.g., thosedescribed in Ausubel et al., supra). For example, a candidate compoundmay be tested in vitro for interaction and binding with a polypeptide ofthe invention and its ability to modulate biofilm formation may beassayed by any standard assay (e.g., those described herein).

In one particular working example, a candidate compound that binds to apolypeptide (e.g, PvrR) may be identified using a chromatography-basedtechnique. For example, a recombinant polypeptide of the invention maybe purified by standard techniques from cells engineered to express thepolypeptide (e.g., those described above) and may be immobilized on acolumn. A solution of candidate compounds is then passed through thecolumn, and a compound specific for the pathogenicity polypeptide (e.g,biofilm regulator polypeptide) is identified on the basis of its abilityto bind to the pathogenicity polypeptide (e.g, biofilm regulatorpolypeptide) and be immobilized on the column. To isolate the compound,the column is washed to remove non-specifically bound molecules, and thecompound of interest is then released from the column and collected.Compounds isolated by this method (or any other appropriate method) may,if desired, be further purified (e.g., by high performance liquidchromatography). In addition, these candidate compounds may be testedfor their ability to render a pathogen incapable of forming a biofilm(e.g., as described herein). Compounds isolated by this approach mayalso be used, for example, as therapeutics to treat or prevent the onsetof a pathogenic infection, disease, or both. Compounds which areidentified as binding to pathogenicity polypeptides (e.g, biofilmregulator polypeptides) with an affinity constant less than or equal to10 mM are considered particularly useful in the invention.

Potential agonists include organic molecules, peptides, peptidemimetics, polypeptides, and antibodies that bind to a nucleic acidsequence or polypeptide of the invention (e.g, biofilm regulatorpolypeptides) and thereby increase its activity. Potential agonists alsoinclude small molecules that bind to and occupy the binding site of thepolypeptide thereby preventing binding to cellular binding molecules,such that normal biological activity is prevented.

Compounds that decrease only antibiotic resistance of a microbe are alsoidentified by monitoring reversion of bacterial colonies from theantibiotic resistant phenotype to the wild-type susceptible phenotype.In one working example, screens for compounds that increase reversionrate are conducted by plating antibiotic resistant variant bacteria onantibiotic-free media in the presence or absence of a candidatecompound. The plates are then cultured using standard methods. Theplates are then visually inspected for revertants, i.e., bacterialcolonies having a wild-type phenotype. The number of wild-type phenotypebacterial colonies is compared between plates cultured in the presenceor absence of a candidate compound. Compounds that increase the numberof wild-type revertants, relative to the number of wild-type revertantson a control plate without the compound, are taken as useful in theinvention.

Additionally, compounds that decrease antibiotic resistance areidentified by monitoring for an increase in the susceptibility ofbacteria to antibiotics. In yet another working example, compounds thatdecrease antibiotic resistance are identified by plating wild-typebacteria on antibiotic containing plates in the presence or absence of acandidate compound. The plates are cultured using standard methods, andthen visually inspected for bacterial colonies. The number of antibioticresistant bacterial colonies is compared between plates cultured in thepresence or absence of a candidate compound. Compounds that decrease thenumber of antibiotic resistant variant colonies, relative to the numberof antibiotic resistant variant colonies on a control plate without thecompound, are taken as useful in the invention.

In another working example, a gene that regulates biofilm formation isidentified by monitoring its activity or activity of its encodedpolypeptide, when mutated. Bacteria are mutagenized using standardmethods, such as transposon mutagenesis. Mutagenized and wild-typebacteria are then plated on antibiotic containing plates. These platesare cultured using standard methods, and then are visually inspected forthe presence of antibiotic resistant variant bacteria. The number ofantibiotic resistant variant bacterial colonies (e.g., small colonyvariants) is compared between mutagenized bacterial plates and wild-typecontrol plates. This comparison is typically conducted when variantcolonies begin to appear on the wild-type plate. A decrease or increasein the number of antibiotic resistant variant bacterial colonies (e.g.,small colony variants) on a plate containing mutagenized bacteria istaken as an indication of the presence of a genetic mutation in a genethat regulates biofilm formation. The mutated gene is then identifiedaccording to standard methods.

In yet another working example, a gene that regulates biofilm orphenotype-mediated antibiotic resistance is identified as follows. Forexample, a candidate gene (e.g., as part of a genomic library) isintroduced into a variant host cell (e.g., Pseudomonas aeruginosa PA14RSCV). Next, the transformed host cell is monitored for reversion fromthe rough small colony variant phenotype to wild-type. The plates arethen cultured using standard methods and monitored for the appearance ofcolonies with a wild-type phenotype. The number of wild-type phenotypebacterial colonies is then compared between plates containingtransformants and variants carrying the vector alone. An increase in thenumber of wild-type revertants, relative to the number of wild-typerevertants on a control plate with the vector alone, identifies a genethat regulates biofilm formation or phenotype-mediated antibioticresistance. A gene identified using this method is subsequently isolatedusing standard procedures known in the art.

In another working example, small colony phenotypic variants are platedon an appropriate antibiotic medium (for example, those describedherein) in the presence of a candidate compound and reversion towild-type is monitored. Compounds that promote reversion from PA14 RSCVto wild-type are taken as being useful in the invention.

In another working example, a gene that regulates or is involved inphenotype-mediated or biofilm-mediated antibiotic resistance or biofilmformation is identified as follows. Bacteria are mutagenized usingstandard methods, such as transposon mutagenesis. Mutagenized bacteriaare then plated on Trypticase Soy Agar (TSA) plates containingantibiotic. These plates are cultured using standard methods, and theninspected for bacterial growth. A decrease in the number of bacterialcolonies or their absence on a mutagenized plate, relative to a controlplate containing non-mutagenized bacteria identifies the presence of agenetic mutation in a gene that regulates phenotype-mediated orbiofilm-mediated antibiotic resistance and biofilm formation. A geneidentified using this method is subsequently isolated using standardprocedures known in the art.

In another working example, a gene that regulates or is involved inphenotype-mediated or biofilm-mediated antibiotic resistance or biofilmformation is identified as follows. Bacteria are mutagenized usingstandard methods, such as transposon mutagenesis. Mutagenized bacteriaare then transferred to Trypticase Soy Broth (TSB) liquid culture mediacontaining an antibiotic. The bacteria are then cultured using standardmethods, and the cultures are inspected for the presence of bacterialgrowth. Bacterial growth is compared between mutagenized cultures andwild-type control cultures. Bacterial growth can be identified, forexample, by visual inspection, by measuring optical density at 600 nm,or by other standard methods. The inability of a mutant to grow inliquid culture with antibiotics indicates the presence of a geneticmutation in a gene that regulates or is involved in phenotype-mediatedor biofilm-mediated antibiotic resistance and biofilm formation. A geneidentified using this method is subsequently isolated using standardprocedures known in the art.

In another working example, a gene that regulates or is involved inphenotype-mediated or biofilm-mediated antibiotic resistance or biofilmformation is identified as follows. Bacteria are mutagenized usingstandard methods, such as transposon mutagenesis. Mutagenized bacteriaare then plated on TSA plates containing antibiotic. These plates arecultured using standard methods, and then inspected for bacterialgrowth. The inability of a mutant to grow in TSA supplemented withantibiotics is taken as an indication of the presence of a geneticmutation in a gene that regulates or is involved in phenotype-mediatedor biofilm-mediated resistance and biofilm formation. A gene identifiedusing this method is subsequently isolated using standard proceduresknown in the art.

In another working example, a gene that regulates or is involved inphenotype-mediated or biofilm-mediated antibiotic resistance or biofilmformation is identified as follows. Bacteria are mutagenized usingstandard methods, such as transposon mutagenesis. Mutagenized bacteriaare then transferred to liquid culture media TSB containing anantibiotic. The bacteria are then cultured using standard methods, andthe cultures are inspected for the presence of bacterial growth.Bacterial growth is compared between mutagenized cultures and wild-typecontrol cultures. Bacterial growth can be identified, for example, byvisual inspection, by measuring optical density at 600 nm, or by otherstandard methods. The inability of a mutant to grow in liquid culturewith antibiotics indicates the presence of a genetic mutation in a genethat regulates or is involved in phenotype-mediated or biofilm-mediatedantibiotic resistance and biofilm formation. A gene identified usingthis method is subsequently isolated using standard procedures known inthe art.

Each of the DNA sequences provided herein may also be used in thediscovery and development of antipathogenic compounds (e.g.,antibiotics). The encoded protein, upon expression, can be used as atarget for the screening of antibacterial drugs. Additionally, the DNAsequences encoding the amino terminal regions of the encoded protein orShine-Delgarno or other translation facilitating sequences of therespective mRNA can be used to construct antisense sequences to controlthe expression of the coding sequence of interest.

The antagonists and agonists of the invention may be employed, forinstance, to inhibit and treat a variety of bacterial infections, forexample, those involving biofilm formation.

Optionally, compounds identified in any of the above-described assaysmay be confirmed as useful in conferring protection against thedevelopment of a pathogenic infection in any standard animal model(e.g., the mouse-burn assay described herein) and, if successful, may beused as anti-pathogen therapeutics (e.g, antibiotics).

Small molecules of the invention preferably have a molecular weightbelow 2,000 daltons, more preferably between 300 and 1,000 daltons, andmost preferably between 400 and 700 daltons. It is preferred that thesesmall molecules are organic molecules.

Test Compounds and Extracts

In general, compounds capable of reducing pathogenic virulence (e.g.,reducing biofilm formation) are identified from large libraries of bothnatural product or synthetic (or semi-synthetic) extracts or chemicallibraries according to methods known in the art. Those skilled in thefield of drug discovery and development will understand that the precisesource of test extracts or compounds is not critical to the screeningprocedure(s) of the invention. Accordingly, virtually any number ofchemical extracts or compounds can be screened using the methodsdescribed herein. Examples of such extracts or compounds include, butare not limited to, plant-, fungal-, prokaryotic- or animal-basedextracts, fermentation broths, and synthetic compounds, as well asmodification of existing compounds. Numerous methods are also availablefor generating random or directed synthesis (e.g., semi-synthesis ortotal synthesis) of any number of chemical compounds, including, but notlimited to, saccharide-, lipid-, peptide-, and nucleic acid-basedcompounds. Synthetic compound libraries are commercially available fromBrandon Associates (Merrimack, N.H.) and Aldrich Chemical (Milwaukee,Wis.). Alternatively, libraries of natural compounds in the form ofbacterial, fungal, plant, and animal extracts are commercially availablefrom a number of sources, including Biotics (Sussex, UK), Xenova(Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, Fla.),and PharmaMar, U.S.A. (Cambridge, Mass.). In addition, natural andsynthetically produced libraries are produced, if desired, according tomethods known in the art, e.g., by standard extraction and fractionationmethods. Furthermore, if desired, any library or compound is readilymodified using standard chemical, physical, or biochemical methods.

In addition, those skilled in the art of drug discovery and developmentreadily understand that methods for dereplication (e.g., taxonomicdereplication, biological dereplication, and chemical dereplication, orany combination thereof) or the elimination of replicates or repeats ofmaterials already known for their anti-pathogenic activity should beemployed whenever possible.

When a crude extract is found to have an anti-pathogenic oranti-virulence activity, or a binding activity, further fractionation ofthe positive lead extract is necessary to isolate chemical constituentsresponsible for the observed effect. Thus, the goal of the extraction,fractionation, and purification process is the careful characterizationand identification of a chemical entity within the crude extract havinganti-pathogenic activity. Methods of fractionation and purification ofsuch heterogenous extracts are known in the art. If desired, compoundsshown to be useful agents for the treatment of pathogenicity arechemically modified according to methods known in the art.

Pharmaceutical Therapeutics

The invention provides a simple means for identifying compounds(including peptides, small molecule inhibitors, and mimetics) capable ofinhibiting the pathogenicity (e.g., biofilm formation) of a pathogen.Accordingly, a chemical entity discovered to have medicinal value usingthe methods described herein is useful as a drug or as information forstructural modification of existing anti-pathogenic compounds, e.g., byrational drug design. Such methods are useful for screening compoundshaving an effect on a variety of pathogens that form biofilms including,but not limited to, bacteria. Examples of pathogenic bacteria include,without limitation, Aerobacter, Aeromonas, Acinetobacter, Agrobacterium,Bacillus, Bacteroides, Bartonella, Bortella, Brucella,Calymmatobacterium, Campylobacter, Citrobacter, Clostridium,Cornyebacterium, Enterobacter, Enterococcus, Escherichia, Francisella,Haemophilus, Hafnia, Helicobacter, Klebsiella, Legionella, Listeria,Morganella, Moraxella, Proteus, Providencia, Pseudomonas, Salmonella,Serratia, Shigella, Staphylococcus, Streptococcus, Treponema,Xanthomonas, Vibrio, and Yersinia.

For therapeutic uses, the compositions or agents identified using themethods disclosed herein may be administered systemically, for example,formulated in a pharmaceutically-acceptable buffer such as physiologicalsaline. Treatment may be accomplished directly, e.g., by treating theanimal with antagonists which disrupt, suppress, attenuate, orneutralize the biological events associated with a pathogenicitypolypeptide (e.g., a biofilm regulator polypeptide). Preferable routesof administration include, for example, subcutaneous, intravenous,interperitoneally, intramuscular, or intradermal injections whichprovide continuous, sustained levels of the drug in the patient.Treatment of human patients or other animals will be carried out using atherapeutically effective amount of an anti-pathogenic agent in aphysiologically-acceptable carrier. Suitable carriers and theirformulation are described, for example, in Remington's PharmaceuticalSciences by E. W. Martin. The amount of the anti-pathogenic agent (e.g.,an antibiotic) to be administered varies depending upon the manner ofadministration, the age and body weight of the patient, and with thetype of disease and extensiveness of the disease. Generally, amountswill be in the range of those used for other agents used in thetreatment of other microbial diseases, although in certain instanceslower amounts will be needed because of the increased specificity of thecompound. A compound is administered at a dosage that inhibits microbialproliferation (e.g., biofilm formation). If desired, such treatment isalso performed in conjunction with standard antibiotic therapy.

Other Embodiments

In general, the invention includes any nucleic acid sequence which maybe isolated as described herein or which is readily isolated by homologyscreening or PCR amplification using the nucleic acid sequences of theinvention. Also included in the invention are polypeptides which aremodified in ways which do not abolish their pathogenic activity(assayed, for example as described herein). Such changes may includecertain mutations, deletions, insertions, or post-translationalmodifications, or may involve the inclusion of any of the polypeptidesof the invention as one component of a larger fusion protein. Also,included in the invention are polypeptides that have lost theirpathogenicity.

Thus, in other embodiments, the invention includes any protein which issubstantially identical to a polypeptide of the invention. Such homologsinclude other substantially pure naturally-occurring polypeptides aswell as allelic variants; natural mutants; induced mutants; proteinsencoded by DNA that hybridizes to any one of the nucleic acid sequencesof the invention under high stringency conditions or, less preferably,under low stringency conditions (e.g., washing at 2×SSC at 40° C. with aprobe length of at least 40 nucleotides); and proteins specificallybound by antisera of the invention.

The invention further includes analogs of any naturally-occurringpolypeptide of the invention. Analogs can differ from thenaturally-occurring the polypeptide of the invention by amino acidsequence differences, by post-translational modifications, or by both.Analogs of the invention will generally exhibit at least 85%, morepreferably 90%, and most preferably 95% or even 99% identity with all orpart of a naturally-occurring amino acid sequence of the invention. Thelength of sequence comparison is at least 15 amino acid residues,preferably at least 25 amino acid residues, and more preferably morethan 35 amino acid residues. Again, in an exemplary approach todetermining the degree of identity, a BLAST program may be used, with aprobability score between e⁻³ and e⁻¹⁰⁰ indicating a closely relatedsequence. Modifications include in vivo and in vitro chemicalderivatization of polypeptides, e.g., acetylation, carboxylation,phosphorylation, or glycosylation; such modifications may occur duringpolypeptide synthesis or processing or following treatment with isolatedmodifying enzymes. Analogs can also differ from the naturally-occurringpolypeptides of the invention by alterations in primary sequence. Theseinclude genetic variants, both natural and induced (for example,resulting from random mutagenesis by irradiation or exposure toethanemethylsulfate or by site-specific mutagenesis as described inSambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual(2d ed.), CSH Press, 1989, or Ausubel et al., supra). Also included arecyclized peptides, molecules, and analogs which contain residues otherthan L-amino acids, e.g., D-amino acids or non-naturally occurring orsynthetic amino acids.

In addition to full-length polypeptides, the invention also includesfragments of any one of the polypeptides of the invention. As usedherein, the term “fragment,” means at least 5, preferably at least 20contiguous amino acids, preferably at least 30 contiguous amino acids,more preferably at least 50 contiguous amino acids, and most preferablyat least 60 to 80 or more contiguous amino acids. Fragments of theinvention can be generated by methods known to those skilled in the artor may result from normal protein processing (e.g., removal of aminoacids from the nascent polypeptide that are not required for biologicalactivity or removal of amino acids by alternative mRNA splicing oralternative protein processing events).

Furthermore, the invention includes nucleotide sequences that facilitatespecific detection of any of the nucleic acid sequences of theinvention. Thus, for example, nucleic acid sequences described herein orfragments thereof may be used as probes to hybridize to nucleotidesequences by standard hybridization techniques under conventionalconditions. Sequences that hybridize to a nucleic acid sequence codingsequence or its complement are considered useful in the invention.Sequences that hybridize to a coding sequence of a nucleic acid sequenceof the invention or its complement and that encode a polypeptide of theinvention are also considered useful in the invention. As used herein,the term “fragment,” as applied to nucleic acid sequences, means atleast 5 contiguous nucleotides, preferably at least 10 contiguousnucleotides, more preferably at least 20 to 30 contiguous nucleotides,and most preferably at least 40 to 80 or more contiguous nucleotides.Fragments of nucleic acid sequences can be generated by methods known tothose skilled in the art.

The invention further provides a method for inducing an immunologicalresponse in an individual, particularly a human, which includesinoculating the individual with, for example, any of the polypeptides(or a fragment or analog thereof or fusion protein) of the invention toproduce an antibody and/or a T cell immune response to protect theindividual from infection, especially bacterial infection (e.g., aPseudomonas aeruginosa infection). The invention further includes amethod of inducing an immunological response in an individual whichincludes delivering to the individual a nucleic acid vector to directthe expression of a polypeptide described herein (or a fragment orfusion thereof) in order to induce an immunological response.

The invention also includes vaccine compositions including thepolypeptides or nucleic acid sequences of the invention. For example,the polypeptides of the invention may be used as an antigen forvaccination of a host to produce specific antibodies which protectagainst invasion of bacteria. The invention therefore includes a vaccineformulation which includes an immunogenic recombinant polypeptide of theinvention together with a suitable carrier.

The invention further provides compositions (e.g., nucleotide sequenceprobes), polypeptides, antibodies, and methods for the diagnosis of apathogenic condition.

All publications and references, including but not limited to patentsand patent applications, cited in this specification are hereinincorporated by reference in their entirety as if each individualpublication or reference were specifically and individually indicated tobe incorporated by reference herein as being fully set forth. Any patentapplication to which this application claims priority is alsoincorporated by reference herein in its entirety in the manner describedabove for publications and references.

1. An isolated polypeptide comprising an amino acid sequence having atleast 50% identity to the amino acid sequence of PvrR (SEQ ID NO:2),wherein expression of said polypeptide, in a microorganism, affectsphenotype-mediated antibiotic-resistance in said microorganism.
 2. Theisolated polypeptide of claim 1, said polypeptide comprising the aminoacid sequence of PvrR (SEQ ID NO:2).
 3. The isolated polypeptide ofclaim 1, wherein said amino acid sequence consists essentially of theamino acid sequence of PvrR (SEQ ID NO:2) or a fragment thereof.
 4. Anisolated polypeptide fragment of the isolated polypeptide of claim
 1. 5.The isolated polypeptide fragment of claim 4, wherein said polypeptidefragment comprises 200 contiguous amino acids of SEQ ID NO:2.
 6. Anisolated polynucleotide having at least 50% identity to the nucleotidesequence of pvrR (SEQ ID NO:1), wherein expression of saidpolynucleotide, in a microorganism, affects phenotype-mediatedantibiotic-resistance in said microorganism.
 7. The isolatedpolynucleotide of claim 6, said polynucleotide comprising the nucleotidesequence of pvrR (SEQ ID NO:1) or a complement thereof.
 8. The isolatedpolynucleotide of claim 7, said polynucleotide consisting essentially ofthe nucleotide sequence of pvrR (SEQ ID NO:1) or a fragment thereof. 9.A vector comprising the isolated polynucleotide of any one of claims 6,7, or
 8. 10. A host cell comprising the vector of claim
 9. 11. Ascreening method for identifying a compound that modulates geneexpression of a regulator polynucleotide that affects phenotype-mediatedantibiotic-resistance in a microorganism, said method comprising thesteps of: (a) providing a microbial cell comprising a polynucleotidehaving at least 50% identity to the nucleotide sequence of pvrR (SEQ IDNO:1), wherein expression of said polynucleotide, in said microbialcell, affects phenotype-mediated antibiotic-resistance in said microbialcell; (b) contacting said microbial cell with a compound; and (c)comparing the level of gene expression of said polynucleotide in thepresence of said compound with the level of gene expression in theabsence of said compound; wherein a measurable difference in geneexpression indicates that said compound modulates gene expression of aregulator polynucleotide that affects phenotype-mediatedantibiotic-resistance in a microorganism.
 12. The method of claim 11,wherein said screening method identifyies a compound that increasestranscription of said regulator polynucleotide.
 13. The method of claim11, wherein said screening method identifies a compound that decreasestranscription of said regulator polynucleotide.
 14. The method of claim11, wherein said screening method identifies a compound that increasestranslation of an mRNA transcribed from said regulator polynucleotide.15. The method of claim 11, wherein said screening method identifies acompound that decreases translation of an mRNA transcribed from saidregulator polynucleotide.
 16. The method of claim 11, wherein thecompound is a member of a chemical library.
 17. The method of claim 11,wherein said microbial cell belongs to the genus Pseudomonas, Vibrio,Salmonella, or Staphylococcus.
 18. The method of claim 11, wherein saidmicrobial cell is a phenotypic variant having increased biofilmformation.
 19. The method of claim 18, wherein said phenotypic variantis a small colony variant.
 20. The method of claim 19, wherein saidsmall colony variant is a small colony variant of Pseudomonas, Vibrio,Salmonella, or Staphylococcus.
 21. The method of claim 18, wherein saidsmall colony variant is a rough small colony variant.
 22. The method ofclaim 21, wherein said rough small colony variant is Pseudomonas,Vibrio, or Salmonella.
 23. The method of claim 11, wherein the activityof the compound is dependent upon the presence of the pvrR gene (SEQ IDNO:1) or a functional equivalent thereof.
 24. The method of claim 11,wherein said compound targets the pvrR gene (SEQ ID NO:1) or afunctional equivalent thereof.
 25. The method of claim 11, whereinexpression of said polynucleotide mediates phenotypic switching of saidmicrobial cell in the presence of a high concentration of an antibiotic.26. The method of claim 11, wherein said polypeptide is expressed by theisolated polynucleotide of any one of claims 6, 7, or
 8. 27. A screeningmethod for identifying a compound that modulates an activity of apolypeptide that affects phenotype-mediated antibiotic-resistance in amicroorganism, said method comprising the steps of: (a) providing amicrobial cell expressing a polypeptide having at least 50% identity tothe amino acid sequence of PvrR (SEQ ID NO:2), wherein expression ofsaid polypeptide, in said microbial cell, affects phenotype-mediatedantibiotic-resistance in said microbial cell; (b) contacting saidmicrobial cell with a compound; and (c) comparing an activity of saidpolypeptide in the presence of said compound with said activity in theabsence of said compound; wherein a measurable difference in theactivity indicates that said compound modulates said activity of saidpolypeptide that affects phenotype-mediated antibiotic-resistance in amicroorganism.
 28. The method of claim 27, wherein said screening methodidentifies a compound that increases the activity of said polypeptide.29. The method of claim 27, wherein said screening method identifies acompound that decreases the activity of said polypeptide.
 30. The methodof claim 27, wherein the compound is a member of a chemical library. 31.The method of claim 27, wherein comparing the activity of thepolypeptide involves an immunological assay.
 32. The method of claim 27,wherein said microbial cell belongs to the genus Pseudomonas, Vibrio,Salmonella, or Staphylococcus.
 33. The method of claim 27, wherein saidmicrobial cell is a phenotypic variant having increased biofilmformation.
 34. The method of claim 33, wherein said phenotypic variantis Pseudomonas aeruginosa PA14 RSCV.
 35. The method of claim 27, whereinsaid regulator polypeptide is the isolated polypeptide of claim
 1. 36.The method of claim 27, wherein the activity of the polypeptideregulates phenotypic switching.
 37. The method of claim 27, wherein theactivity of the polypeptide regulates biofilm-mediatedantibiotic-resistance.
 38. The method of claim 27, wherein the activityof the polypeptide affects susceptibility of the microbial cell toantibiotic treatment.
 39. The method of claim 27, wherein saidpolypeptide is an element of a two-component regulatory system.
 40. Themethod of claim 27, wherein the activity of the compound is dependentupon the presence of the PvrR polypeptide (SEQ ID NO:2) or a functionalequivalent thereof.
 41. The method of claim 27, wherein said compoundtargets the PvrR polypeptide (SEQ ID NO:2) or a functional equivalentthereof.
 42. The method of claim 27, wherein said polypeptide mediatesphenotypic switching of said microbial cell in the presence of a highconcentration of an antibiotic.
 43. The method of claim 27, wherein saidpolypeptide is expressed by the isolated polynucleotide of any one ofclaims 6, 7, or
 8. 44. A screening method for identifying a compoundthat modulates microbial biofilm formation, said method comprising thesteps of: (a) culturing a microbial cell comprising a polypeptide havingat least 50% identity to the amino acid sequence of PvrR (SEQ ID NO:2),wherein said microbial cell, upon culturing, forms a biofilm; (b)contacting said microbial cell with a compound; and (c) comparingmicrobial biofilm formation in the presence of said compound withmicrobial biofilm formation in the absence of said compound; wherein ameasurable difference in said microbial biofilm formation indicates thatsaid compound modulates biofilm formation.
 45. The method of claim 44,wherein said screening method identifies a compound that increasesbiofilm formation.
 46. The method of claim 44, wherein said screeningmethod identifies a compound that decreases biofilm formation.
 47. Themethod of claim 44, wherein biofilm formation is measured by assayingmicrobial aggregation.
 48. The method of claim 47, wherein microbialaggregation is assayed using a microscope.
 49. The method of claim 47,wherein microbial aggregation is assayed using a salt aggregation test.50. The method of claim 47, wherein microbial aggregation is assayedusing an attachment assay.
 51. The method of claim 44, wherein thecompound is a member of a chemical library.
 52. The method of claim 44,wherein said microbial cell belongs to the genus Pseudomonas, Vibrio,Salmonella, or Staphylococcus.
 53. The method of claim 44, wherein saidmicrobial cell is a phenotypic variant having increased biofilmformation.
 54. The method of claim 53, wherein said phenotypic variantis a small colony variant.
 55. The method of claim 54, wherein saidsmall colony variant is a small colony variant of Pseudomonas, Vibrio,Salmonella, or Staphylococcus.
 56. The method of claim 54, wherein saidsmall colony variant is a rough small colony variant.
 57. The method ofclaim 56, wherein said rough small colony variant is Pseudomonas,Vibrio, or Salmonella.
 58. The method of claim 44, wherein the activityof the compound is dependent upon the presence of PvrR polypeptide (SEQID NO: 2) or a functional equivalent thereof.
 59. The method of claim44, wherein said compound targets the PvrR polypeptide (SEQ ID NO:2) ora functional equivalent thereof.
 60. The method of claim 44, whereinexpression of said polypeptide mediates phenotypic switching of saidmicrobial cell in the presence of a high concentration of an antibiotic.61. The method of claim 44, wherein said polypeptide is an isolatedpolypeptide of any one of claims 1, 2, or
 3. 62. A method of treating amicrobial infection involving a microorganism that forms a biofilm in amammal, said method comprising administering to said mammal atherapeutically-effective amount of a compound that induces theexpression of or activity of or represses the expression of or activityof the polypeptide of any one of claims 1, 2, or
 3. 63. The method ofclaim 62, wherein said method further comprises administering to saidmammal a therapeutically-effective amount of an antibiotic.
 64. Themethod of claim 62, wherein said mammal is a human.
 65. The method ofclaim 62, wherein said human has cystic fibrosis.
 66. The method ofclaim 62, wherein said human has a chronic infection.
 67. The method ofclaim 62, wherein the said microorganism belongs to the genusPseudomonas, Vibrio, Salmonella or Staphylococcus.
 68. A method ofcleaning or disinfecting a surface at least partially covered by amicroorganism that forms a biofilm, said method comprising contactingsaid microorganism with a cleaning composition comprising a compoundthat induces the expression of or activity of or represses theexpression of or activity of the polypeptide of claim 1, 2, or
 3. 69.The method of claim 68, wherein said microorganism belongs to the generaPseudomonas, Vibrio, Salmonella or Staphylococcus.
 70. A screeningmethod for identifying a compound that decreases pathogenicity of anantibiotic-resistant phenotypic variant, said method comprising thesteps of: (a) contacting an antibiotic-resistant phenotypic variant witha candidate compound; and (b) measuring reversion of saidantibiotic-resistant phenotypic variant to a wild-type phenotype, anincrease in reversion indicating that said compound decreasespathogenicity of said antibiotic-resistant phenotypic variant.
 71. Themethod of claim 70, wherein said antibiotic-resistant phenotypic variantis a bacterial variant.
 72. The method of claim 71, wherein saidantibiotic-resistant phenotypic bacterial variant is cultured in theabsence of an antibiotic.
 73. The method of claim 71, wherein saidantibiotic-resistant phenotypic bacterial variant has increased biofilmformation.
 74. The method of claim 71, wherein said antibiotic-resistantphenotypic bacterial variant is a rough small colony variant.
 75. Themethod of claim 71, wherein said antibiotic-resistant phenotypicbacterial variant is a hyperpiliated variant.
 76. The method of claim71, wherein said antibiotic-resistant phenotypic bacterial variant hasincreased hydrophobicity.
 77. The method of claim 71, wherein saidantibiotic-resistant phenotypic bacterial variant has an alteration in asurface component.
 78. The method of claim 71, wherein saidantibiotic-resistant phenotypic bacterial variant is a pathogen.
 79. Themethod of claim 78, wherein said pathogen is a Gram positive bacterium.80. The method of claim 79, wherein said pathogen is Staphylococcus. 81.The method of claim 78, wherein said pathogen is a Gram negativebacterium.
 82. The method of claim 75, wherein said pathogen is Vibrio,Pseudomonas, or Salmonella.
 83. A screening method for identifying acompound that decreases pathogenicity of a wild-type microbe, saidmethod comprising the steps of: (a) culturing a wild-type microbe with acandidate compound in the presence of an antibiotic; and (b) comparingthe number of antibiotic-resistant phenotypic variants in the presenceof said compound to the number of antibiotic-resistant phenotypicvariants in the absence of said compound, a decrease in the number ofsaid antibiotic-resistant phenotypic variants in the presence of saidcompound indicating that said compound decreases pathogenicity of saidwild-type microbe.
 84. A screening method for identifying apolynucleotide encoding a regulator polypeptide that modulates anantibiotic-resistant phenotype of a microorganism, said methodcomprising the steps of: (a) identifying an antibiotic-resistantphenotypic variant of a microorganism comprising a first phenotype; (b)mutagenizing said antibiotic-resistant phenotypic variant of saidmicroorganism, thereby generating a mutated phenotypic variant of saidmicroorganism; and (c) selecting said mutated phenotypic variant of step(b) having a second phenotype, other than the first phenotype of saidantibiotic-resistant phenotypic variant, wherein said second phenotypeidentifies a mutation in said mutated phenotypic variant of step (b);and (d) using said mutation for identifying a polynucleotide encoding aregulator polypeptide that modulates an antibiotic-resistant phenotypeof a microorganism.
 85. The method of claim 84, wherein said secondphenotype comprises a wild-type phenotype.
 86. A screening method foridentifying a polynucleotide encoding a regulator polypeptide thatmodulates phenotype-mediated antibiotic-resistance of a microorganism,said method comprising the steps of: (a) transforming anantibiotic-resistant phenotypic variant of a microorganism with acandidate polynucleotide encoding a regulator polypeptide; and (b)culturing said transformed antibiotic-resistant phenotypic variant of amicroorganism under conditions suitable for expression of said regulatorpolypeptide; and (c) measuring reversion of said transformedantibiotic-resistant phenotypic variant of said microorganism to awild-type phenotype, an increase in reversion identifies saidpolynucleotide as encoding a regulator polypeptide that modulatesphenotype-mediated antibiotic-resistance.
 87. The method of claim 80,wherein said polynucleotide encodes a regulator polypeptide thatmodulates a phenotypic switch from antibiotic-resistant phenotype to anantibiotic-susceptible phenotype.
 88. The method of claim 80, whereinsaid polynucleotide having at least 50% identity to the nucleotidesequence of pvrR (SEQ ID NO:1) encodes an element of a two-componentregulatory system.